Preface

This book presents the latest research and treatment options in cartilage disorders. It provides an in-depth understanding of the cutting-edge research, technologies, and treatment options currently available to orthopedic surgeons.

Over the past few decades, there have been significant advances in our understanding of the pathophysiology of cartilage disorders as well as in the development of new treatment options for these conditions. Cartilage disorders are complex and multifactorial, and they can cause significant pain and disability in affected individuals. It is therefore essential to have a thorough understanding of the latest research and treatment options to provide the best possible care to patients.

Written by leading experts in cartilage research, the chapters in this textbook provide an in-depth analysis of the latest research findings and treatment options. The book is divided into two sections. Section I provides an overview of the basic science of cartilage biology, including the structure and function of cartilage, as well as the molecular mechanisms that underlie cartilage disorders. Section II focuses on the clinical aspects of cartilage disorders, including the regenerative techniques and treatment of these conditions.

The chapters in Section I cover topics such as the structure and composition of cartilage, the molecular mechanisms of cartilage degeneration, and the role of inflammation in cartilage disorders. The authors also discuss the latest advances in imaging and biomarker technologies, which have greatly enhanced our ability to diagnose and monitor cartilage disorders.

Section II of the book focuses on the clinical aspects of cartilage disorders, including the evaluation and management of patients with cartilage injuries. The authors provide detailed descriptions of the various techniques used to repair and regenerate damaged cartilage, as well as the latest advances in cell-based therapies and tissue engineering approaches. The association of cartilage lesions and ACL injuries is also highlighted in depth.

The editors of this textbook have taken great care to ensure that the content is both informative and accessible to specialized orthopedic surgeons. The chapters are written in a clear and concise style, and they are accompanied by numerous illustrations and diagrams to aid in understanding. In addition, each chapter includes a comprehensive reference list to facilitate further reading and research.

In conclusion, this textbook represents a comprehensive and up-to-date resource for specialists who are interested in the latest research and treatment options in cartilage disorders. We hope that readers will find this book to be a valuable addition to their professional library and that it will enhance their ability to provide the best possible care to their patients.

We would like to thank our families for their support. Ahmed dedicates this book to his parents, loving wife, daughter, and son. Taiceer dedicates this textbook to his loving parents, brother Omer, sister Noor, and nieces Yasmine and Noora.

And of course, our utmost dedication to our dear friend, role model, the true professor, Prof. Karl Fredrik Almqvist. He has trained us, guided us, and supported us in the operating theatre, the clinic, and life.

> **Karl Almqvist** Orthocure Medical Center, Dubai, UAE

**Taiceer Abdulwahab and Ahmed Ebrahim El Hamaky**

Mediclinic City Hospital, Dubai, UAE

**1**

Section 1

Cartilage Degeneration

and Biology

Mohammed Bin Rashid University of Medicine and Health Sciences (MBRU), Dubai, UAE

Section 1

## Cartilage Degeneration and Biology

#### **Chapter 1**

## Introductory Chapter: Cartilage Disorders

*Taiceer Abdulwahab*

#### **1. Introduction**

This book demonstrates the pathological changes of cartilage degeneration at the molecular level and during the early stages of osteoarthritis, biochemical changes that precede the later structural changes.

Hyaline Articular Cartilage is critical for the proper functioning of the joint as it provides a smooth and low-friction surface for bone movement. However, hyaline cartilage has a limited ability to repair itself, making it vulnerable to damage and degeneration. In recent years, researchers have developed various techniques for the regeneration of hyaline cartilage, including Confocal Raman Microscopy for identifying tissue damage, tissue engineering for cartilage regeneration and autologous chondral implantations.

#### **2. Confocal Raman microscopy**

Confocal Raman Microscopy (CRM) is a promising tool for the characterisation of the collagen and proteoglycan content, as well as for monitoring the tissue damage and repair [1]. CRM can detect subtle changes in the Extra-Cellular Matrix (ECM) of articular cartilage, such as variations in collagen cross-linking, proteoglycan content and mineralisation. Thus, CRM has the potential to be used as a non-invasive diagnostic tool for early detection and monitoring of cartilage degeneration and repair.

#### **3. Tissue engineering for cartilage regeneration**

Tissue engineering approaches for cartilage regeneration aim to overcome the limitations of current treatments by providing functional and long-lasting repair tissue [2]. The most commonly used biomaterials for cartilage tissue engineering are hydrogels, which are highly hydrated and have a similar composition to the native ECM [3]. Hydrogels can provide mechanical support and act as a template for cell growth, differentiation and matrix deposition. They can also be modified with bioactive molecules, such as growth factors and cytokines, to promote cell proliferation and matrix synthesis [4].

#### **4. Autologous chondral implantation**

Autologous Chondral Implantation (ACI) is a cell-based therapy that involves harvesting healthy chondrocytes from a non-weight-bearing area of the patient's joint and growing them in vitro. The expanded chondrocytes are then implanted into the damaged area of the joint using a periosteal or collagen membrane. ACI has shown promising results in the treatment of osteochondral defects, especially in the knee joint, with a reported success rate of 75–90% [5].

#### **5. Conclusion**

In conclusion, cartilage is a highly specialised tissue that is critical for joint function, but its limited self-repair capacity and the prevalence of joint disorders have created a critical need for advanced cartilage repair therapies. Confocal Raman microscopy, tissue engineering and autologous chondral implantation are among the latest scientific methods that show promise in advancing the field of cartilage regeneration. Although these approaches have shown significant progress, further research is needed to optimise their efficacy and long-term outcomes [6].

We endeavour that this book will update the current Orthopaedic Sports Surgeon on these advances in technique and technology, with explorations of future research and technique modalities.

#### **Author details**

Taiceer Abdulwahab1,2

1 Mediclinic City Hospital, Dubai, UAE

2 Mohammed Bin Rashid University of Medicine and Health Sciences (MBRU), Dubai, UAE

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Introductory Chapter: Cartilage Disorders DOI: http://dx.doi.org/10.5772/intechopen.111782*

#### **References**

[1] Marcolongo M, Ateshian GA. Confocal Raman microscopy for cartilage characterization and monitoring. Current Opinion in Biomedical Engineering. 2018;**6**:43-50

[2] Liu Y, Wu X, Liao Y, Zhou N, Wu F, Cui L, et al. Advances and challenges in biomaterials for articular cartilage tissue engineering. Acta Biomaterials. 2020;**112**:1-26

[3] Ng KW, Wang CC, Mauck RL, Kelly TA, Chahine NO, Costa KD, et al. A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-laden hydrogel constructs. Biomaterials. 2011;**32**(29):7419-7426

[4] Toh WS, Lim TC, Kurisawa M, Spector M. Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials. 2012;**33**(2):383-391

[5] Giannini S, Buda R, Vannini F, Di Caprio F, Grigolo B. Autologous chondrocyte transplantation in osteochondral lesions of the ankle joint. Foot and Ankle Clinics. 2001;**22**(6):513-517

[6] Hunziker EB. Articular cartilage repair: Are the intrinsic biological constraints undermining this process insuperable? Osteoarthritis and Cartilage. 1999;**7**(1):15-28

#### **Chapter 2**

## Characterization of Degraded Cartilage Using Confocal Raman Microscopy

*N'Dre Jean, Hamideh Salehi, Marie Maumus, Danièle Noël, Yolande Koffi-Gnagne and Frédéric Cuisinier*

#### **Abstract**

Osteoarthritis is a degenerative disease with pathological changes at the molecular level. Moreover, the damage to articular cartilage is irreversible. Early detection and the ability to follow the progression of osteoarthritis are essential to anticipate management. To characterize degraded human articular cartilage and to identify cellular changes that are precursors of phenotypic matrix changes in osteoarthritis, normal and degraded articular cartilage explants were harvested from the same patient's knee after informed consent. The blocks were washed several times (four times) with phosphate-buffered saline (often abbreviated to PBS) and then fixed on CaF2 slides using Cell-Tak® (an adhesive glue), and the whole set was placed in different Petri dishes containing PBS for Raman measurements. The analysis of the spectroscopic data allowed to differentiate degraded cartilage from normal cartilage by applying intensity ratios of some Raman bands and/or spectral regions. In addition, peaks at 864, 929, 945, 1107, 1386, and 2887 cm−1 were identified as characteristic Raman markers of degraded cartilage. The use of confocal Raman microscopy (CRM) has proven to be relevant in providing biochemical information necessary to characterize OA cartilage. CRM appears to be a powerful tool for the diagnosis and therapeutic evaluation of osteoarthritis in both early and late stages.

**Keywords:** articular cartilage, osteoarthritis, confocal Raman microscopy, degraded cartilage, Raman spectral data

#### **1. Introduction**

Osteoarthritis (OA) is the most common degenerative joint disease and one of the main causes of morbidity and economic burden for health resources. It is a slowly progressive disease that alters all tissues of the affected joint, with a long asymptomatic period [1]. According to OARSI (International Association for the Study of Osteoarthritis), OA is a serious disease defined as a disorder involving mobile joints, characterized by adhesive stress and degradation of the extracellular matrix, resulting in macro- and micro-damage that activates abnormal adaptive restorative responses, including pro-inflammatory pathways of the immune system [2]. Emerging evidence

in recent years defines OA as a heterogeneous, multifaceted disease with multiple molecular and clinical phenotypes [3, 4]. Loss of articular cartilage structure and function is one of the main features of OA [5–7].

The current diagnosis of OA is based primarily on radiographic criteria (e.g., joint space width, osteophyte formation, subchondral sclerosis) and clinical symptoms (e.g., pain, stiffness, and loss of function). Radiography is the most accessible tool for assessing OA: It can show lesions and other changes related to OA to confirm its severity according to different classification systems, such as Kellgren's Lawrence classification system [8]. MRI, which does not use radiation, is more expensive than X-rays but can provide better images of cartilage and other structures to detect early abnormalities in OA [9]. Another imaging technique is optical coherence tomography (OCT) imaging, which has the ability to generate cross-sectional images of articular cartilage and can provide quantitative information about the condition of articular cartilage, particularly for OA caused by changes in collagen structure [10]. Although these different medical tests are more sensitive than plain radiography, they cannot be routinely applied to many patients due to its cost, and if so, they are often time-consuming and even destructive. The other disadvantage is that these techniques are only valid and feasible in the advanced stages of osteoarthritis.

Currently, the lack of validated biomarkers and early diagnostic tools is one of the major obstacles to improved diagnosis and therapeutic evaluation of OA [11]. Recently, Raman spectroscopic techniques have been shown to not only provide noninvasive and nondestructive structural information in damaged cartilage, but also to allow spatial resolution at the biomolecular level that can be useful in detailed structural analyses of cartilage diseases. Indeed, these techniques can identify functional groups and chemical bonds present in biological tissues and/or cells. As a result, it is possible not only to assess the structure of proteins, lipids, carbohydrates, and nucleic acids present in a biological molecule [12–15], but also the changes in their chemical structure due to the disease process [3, 11], thus allowing monitoring of the progression of the disease process and prediction of the chemical pathway of the progression. Vibrational spectroscopy thus appears to be a proven analytical tool for understanding chemical changes associated with pathological conditions in tissues. The objective of our study was to characterize degraded human articular cartilage using confocal Raman microscopy (CRM) and to identify early cellular changes that are precursors to the phenotypic change of the matrix in OA.

#### **2. Materials and methods**

#### **2.1 Source and preparation of samples**

Our samples consisted of human articular cartilage explants taken from the knee of one patient (**Figure 1**). The biopsies were taken from the same knee but at different locations. For example, degraded cartilage (test sample) at the lesion site and normal cartilage (control sample) away from the lesion site. Human tissue was obtained for research purposes with donor consent. The study was also approved for the recovery of OA samples by the Ministry of Research and Innovation and the Comité d'Éthique de la Protection des Données Personnelles (CPP) of Languedoc-Roussillon (approval DC-2010-1185). The cartilage samples were collected from the same patient. The collected sample blocks were washed several times (four times) with phosphate-buffered *Characterization of Degraded Cartilage Using Confocal Raman Microscopy DOI: http://dx.doi.org/10.5772/intechopen.107310*

**Figure 1.** *Cartilage samples taken from a patient's knee.*

saline (often abbreviated to PBS) and then fixed on CaF2 slides using Cell-Tak® (adhesive glue). The whole set was placed in different Petri dishes containing PBS for Raman measurements. During the whole measurement period, the samples were kept in a refrigerator at 4°C.

#### **2.2 Raman measurements of the samples**

All measurements were performed using a Witec α 300R confocal Raman microscope (Witec, Ulm, Germany) (**Figure 2a**). The system was equipped with a dual-frequency Nd: YAG laser (Newport, Evry, France) with a wavelength of 532 nm and a NIKON × 20 aerial lens with a numerical NA of 0.46 (Nikon, Tokyo, Japan). The output laser power was 50 mW. The spatial resolution was 300 nm, and the depth resolution was approximately 1 μm. The microscope was equipped with a piezo-driven scanning stage with a positioning accuracy of 2–3 nm horizontally and 10 nm vertically, respectively. The acquisition time for a single spectrum was set to 0.5 s; 150 × 150 points per image were recorded, resulting in a total of 22,500 spectra for one image. Data acquisition was performed using Image Plus 2.08 software from Witec. Using an edge filter, the backscattered Raman radiation was

#### **Figure 2.**

*Measurement of articular cartilage using confocal Raman microscopy. (a) Confocal Raman microscope at Laboratoire Bioingénierie et nanoscience UM\_104 Montpellier. (b) Schematic representation of the system. The laser light source irradiates the articular cartilage, and the scattering light is generated by the scattering of the samples. The Raman scattering light is obtained by the filter, and the Raman spectrum is presented after its detection and processing by a CCD detector. The data of the spectra are analyzed using multivariate or chemometric methods.*

separated from the scattered Rayleigh light. The Raman photons were transferred to the EMCCD camera (DU 970 N-BV353, Andor, Hartford, USA). The EMCCD chip size is 1600 × 200 pixels, the camera controller is a 16-bit A/D converter operating at 2.5 MHz, and the camera is cooled by a Pelletier system. The UHTS 300 spectroscopy system with 70% transmission and a 600 lines per mm grating (operating at \_ 60° C) provides a spectral resolution of 3–5 cm−1 . This microscope was used for the analysis of articular cartilage structures.

#### **2.3 Analysis of the Raman spectral data**

All collected spectra were preprocessed in order to obtain spectra that not only have the same scale, but are comparable to each other. The spectral analysis was performed in the fingerprint range (600–1800 cm−1 ) due to its higher molecular specificity. Prior to the multivariate statistical analysis, the Raman spectra were preprocessed using well-established techniques. The preprocessing process of the spectra consisted, first, of baseline subtraction following the eighth-order polynomial law. Subsequently, we proceeded to the elimination of the autofluorescence of the tissues and the smoothing of the spectra using the "Savitzky Galay" filter, following a polynomial order = 4 with a number of points (or interval = 13). In order to compare the spectra and allow consistent comparisons where intensity variations may be relative to the intensity of each spectrum, the data were normalized to the area of the region between 600 and 1800 cm−1 [15]. These steps of preprocessing the spectra were necessary before subjecting them to statistical methods. All preprocessing, normalization, and determination of the different intensities of the peaks or bands were performed with the non-commercial Spectragryph® software version 1.2.12 developed and kindly offered by Dr. Friedrich Menges.

In addition, multivariate analyses such as principal component analysis (PCA) were applied to the raw dataset collected from the different samples. PCA is a well-established multivariate data analysis method that is well suited to distinguish small recurrent spectral variations from large datasets containing uncorrelated variations. It is a completely unsupervised analysis method for establishing whether or not sample spectra are grouped into classes based on sample type among other factors. This method greatly reduces the size of the dataset into a defined number of principal components (PCs), as all spectra are expressed in terms of a few basic functions (usually <10) and "a score vector" of about p entries. Thus, if clustering is observed, there are quantifiable and significant variations in the spectra that can be used to build discriminative algorithms to distinguish between different samples. PCA describes large global changes in composition without any prior knowledge.

#### **3. Statistical analysis**

Given that the distribution of our data does not follow a normal distribution, all our statistical analyses were performed using a non-parametric test, namely the Kruskal-Wallis one-way analysis of Variance on Ranks statistical test with p < 0.001 for significance of our results. For comparisons between different areas, the Student-Newman-Keuls statistical test with p-value <0.05 was applied. All our data were processed with SigmaPlot for Windows software version 11.0 Build <IP_ADDRESS>.

### **4. Results**

#### **4.1 Characterization of articular cartilage degradation**

From the collected cartilage blocks, a total of 50 Raman spectra were collected at different locations on each normal and degraded cartilage sample. The degraded state of the cartilage was assessed by determining the content of PGs and type II collagen, the main components of the extracellular matrix of cartilage, as reported in previous studies [16]. In **Figure 3**, we can easily see that the content of PGs significantly decreased (p < 0.001) in degraded cartilage, with a random coil content, characterized by applying the ratios of the integrated areas of the carbohydrate region ranging from 985 to 1185 cm−1 to the amide I region (1601–1776 cm−1 ) [16], the intensity ratio of the two amide III peaks (1241/1269 cm−1 ), with the peak 1241 cm−1 corresponding to the random coil content = NH2 bending: Random coil Amide III and 1269 cm−1 corresponding to the alpha-helix content = NH2 bending: alpha-helix Amide III) [17, 18], respectively.

#### **4.2 Multivariate analysis and discrimination of Raman bands involved in articular cartilage degradation**

The application of PCA on the raw spectra of the different samples allowed us to discriminate degraded cartilage from normal cartilage. **Figure 4** shows us the average and differential Raman spectra with the characteristic differential Raman bands. Also, analyzing the PCA score plot using CP1 and CP2, as well as that using CP2 and CP3, we find that only CP2 separates the degraded/normal samples (**Figure 5**). The CP2 principal component shows the intense positive charges for the Raman shifts in the degraded cartilage, characterized by the localized peaks, respectively at: 788 cm−1 ( \_ O\_ P\_ O \_ ); at 821 cm−1 (\_ O\_ P\_ O \_ ); 867 cm−1 (RNA); 886 cm−1 (collagen I); 926 cm−1 (ν (C-C)); 945 cm−1 (ν (C-C) backbone); 972 cm−1 (ν (C-C) backbone in RNA); 1107 cm−1 (ν (C-O)); 1274 cm−1 (Amide III); 1386 cm−1 (GAG); 1432 cm−1 (CH2 scissoring); 1480 cm−1 (CH deformation); 2887 cm−1 (CH2 stretch); and 2947 cm−1 (ʋas CH2, lipids, fatty acids). These peaks are the expression of vibrations of nucleic

#### **Figure 3.**

*Characterization of degraded cartilage: (A) determination of PGs content by the ratio of the area of the carbohydrate region [985–1185]/amid region I [1601–1760]. The histogram shows a higher intensity ratio for normal cartilage (CA). This reflects a significant (p < 0.001) decrease in PGs in degraded cartilage. (B) The ratio of disordered collagen (random coil) to ordered collagen (α-helix), represented by the intensity ratio I1251/I1271, is significantly greater (p < 0.001) and in favor of the random coil in degraded cartilage. Observations: D: Degraded cartilage; N: Normal cartilage.*

**Figure 4.**

*Average and differential Raman spectra (n = 50 spectra).*

#### **Figure 5.**

*Principal component analysis. (A) PCA score plot using PC1 and PC2. (B) PCA score plot using PC2 and PC3. Only PC2 separates degraded/normal samples. (C) PC2 loading is responsible for separating degraded and normal cartilage samples. (D) Remaining PC1 and PC3 loadings.*

acid bases, collagen, and GAGs. In contrast, the negative charge peaks are the characteristic Raman shifts in normal cartilage, reflected in particular by peaks at 1004 cm−1 (Phe), 1624 cm−1 (Trp), 1683 cm−1 (ν (C=O)), and 2933 cm−1 (CH2 asymmetric stretch). The assignments of these different peaks are recorded in **Table 1**.

#### **5. Discussion**

Pathological changes in cartilage begin at the molecular level (at the nanoscale) from where they propagate to higher levels of the hierarchical cartilage architecture

#### *Characterization of Degraded Cartilage Using Confocal Raman Microscopy DOI: http://dx.doi.org/10.5772/intechopen.107310*

#### **Table 1.**

*Table of major Raman peak or band assignments involved in the characterization of degraded versus non-degraded (normal) articular cartilage [12, 17, 19–21].*

and cause increasingly irreversible structural and functional damage. Although the etiology of osteoarthritis is largely unknown, its onset is characterized by an imbalance between catabolic and anabolic processes, promoting the degradation of the cartilage matrix. The underlying cause of these macroscopic and microscopic structural features has been linked to the change in biochemical compositions such as alteration and decrease in proteoglycan content but also by the disorganization of type II collagen fibers [18, 22]. The biochemical changes are triggered by the expression of enzymes that are responsible for matrix degradation. The main components of articular cartilage are GAGs (15–30% of dry weight) and collagen (50–60% of dry weight) [23, 24], and their changes can be used as indicators for the early diagnosis of OA. Therefore, any technique to diagnose the onset of OA must detect early changes in cartilage PG gel before significant changes in its collagen matrix occur.

Raman spectroscopy has been shown to provide information on protein structure. Indeed, subtle molecular changes often cause detectable vibrational changes that can be detected by Raman analysis. In recent studies, Raman has been used either to more accurately quantify the distribution of cartilage subcomponents over its entire surface [25] or in some situations to establish the difference between different regions of the tissue over its entire depth the tissue surface [26].

Thus, Raman spectroscopy can be useful in differentiating normal from degraded cartilage. It is now known that the intensity ratio of the two peaks (I1251/I1271 cm−1 ) provides information about the protein structure [11, 17]. Still called the ratio of

disordered collagen (random coil) to ordered collagen (α-helix), represented by the intensity ratio 1241/1269 cm−1 , provides an appreciation of articular cartilage damage [27]. This intensity ratio increases with the progression of osteoarticular diseases following the degree of cartilage damage (ICRS). In the present case, **Figure 3** shows a higher intensity ratio (1251/1271 cm−1 ) and thus in favor of disordered collagen, characterizing a disordered structure of the protein. This result indicates an increase in defective collagen content. This observation was also made by Kumar et al. [18], who, when analyzing different grades of osteoarthritis, showed that this intensity ratio increased the progression of the cartilage disorder. This alteration of the random coil is associated with a decrease in PGs marking a clear difference between normal and degraded cartilage.

Other more subtle changes were highlighted by the application of PCA, which is a common multivariate statistical method very often used in bioanalytical Raman spectroscopy to reduce dimensionality and identify combinations of the most important spectral markers that maximize data variance and optimize group separation [18]. Indeed, although univariate analysis of Raman intensities in normalized spectra was necessary to differentiate degraded cartilage from normal cartilage by determining proteoglycan and type II collagen contents, multivariate analysis, particularly PCA, has been shown to be very effective in discriminating the two samples and identifying characteristic Raman bands or peaks. This ability of PCA has been widely demonstrated in numerous tissue and cell Raman spectroscopy studies [18, 25].

**Figure 5C** shows the loadings of the Raman bands responsible for the separation of the degraded and normal cartilage samples. Focusing on the peaks of the degraded cartilage, we find a high nucleotide activity (expressed by the presence of bands at 788, 821, and 1480 cm−1 ), which could be attributed to the increased internucleosomal DNA cleavage manifested in the advanced stages of OA as revealed also by Verrier et al. [28]. During the progression of ECM degradation, chondrocytes are strongly solicited to renew the matrix proteins subjected to degradation phenomena. This high level of activity can lead to the death of chondrocyte cells by exhaustion, as demonstrated in the work of Zamli et al. [29] after evaluating the role of chondrocyte apoptosis in spontaneous animal models of osteoarthritis. They further demonstrated that chondrocyte death correlated with CA fibrillation (r = 0.3), cellularity< (r = 0.4), proteoglycan depletion, and overall OA microscopic scores (r = 0.4). This supports our results with the depletion of PGs and alteration of type II collagen structure observed in degraded cartilage and characterized by the presence of the high Raman signal intensity peaks at 867, 886, 926, 945 cm−1 , and 1274 cm−1 (collagen attribution) and peaks at 1107, 1342, and 1386 cm−1 (GAGs attribution). Focusing on the 1274 cm−1 peak, amide III attribution, Shaikh et al. [30] recently showed significant variations related to the intensity of this band and δCH2 stretching in a group of cartilage with impact injuries. They therefore inferred that these changes in the intensity of the amide III peak were potentially due to conformational and configurational changes in collagen macromolecules as well as proteoglycan depletion. A first study by Lim et al. [31] had already shown, after an impact on porcine cartilage, a red shift of the 1264–1274 cm−1 peak. They correlated this shift of amide III with the compression of the C-N vibration in the collagen fibers. In the case of our study, the presence of the 1274 cm−1 peak could therefore reflect an alteration of the random coil associated with the decrease of the PGs content observed above. In addition, the presence of the peak at 1386 cm−1 , corresponding to N-acetyl-glucosamine (attribution of hyaluronic acid (HA)), helps to corroborate our observation. It is known that HA constitutes the main skeleton of GAGs. This glycoprotein binds to other aggrecanes to

#### *Characterization of Degraded Cartilage Using Confocal Raman Microscopy DOI: http://dx.doi.org/10.5772/intechopen.107310*

form superaggregates responsible for tissue hydration, allowing cartilage to respond to mechanical stress. Thus, it helps to organize and stabilize the relationship between PGs and the collagen meshwork. Any changes in this glycoprotein could necessarily lead to disruptions in the structure of the cartilage matrix. As an important component of synovial fluid, HA can increase joint flexibility, thereby reducing cartilage wear. In a study by Buckwalter et al. [32], it could be shown that one of the first changes associated with osteoarthritis and joint immobilization was related to changes in hyaluronic acid glycoprotein. Also, Safiri et al. [33] recently demonstrated that it was associated with the radiological progression of osteoarthritis.

One factor that has also been associated with OA is the appearance of lipid deposits especially in the advanced stages of OA [34]. More recently, Mansfield and Winlove [12] studied the distribution of lipids, identifying two related regions: a band at 1441 cm−1 and shifts from 2845 to 2930 cm−1 . Generally, the CH vibrational region between 2800 and 3000 cm−1 is of particular interest for spectroscopic studies because it provides information about the chemical compositions of the samples due to differences in the C-H bond environment [12, 31]. Thus, it can provide information about the relative total concentration of biomolecules but also about changes in chemical bonds. In a recent study, Gaifulina et al. [35] were able to correlate the alterations observed in cartilage with the loss of tissue constituents and the observed increase in water content. They therefore correlated the observed increase in cartilage hydration with the increase in the intensity of the O-H stretch band. In this study, the Raman shifts of the bands at 2887 and 2947 cm−1 (lipid and/or fatty acid vibration) could reflect the strong presence of lipids also in degraded cartilage and explain the possible changes within the cytoplasmic skeleton by lipid production in the face of OA progression. This is justified by the response of chondrocytes to cartilage degradation in order to cope with the physiochemical changes in the cartilage matrix. It should be remembered that lipids are important nutrients in the metabolism of chondrocytes and are available to these cells by *de novo* synthesis, but also by diffusion from the surrounding tissues. Amanda et al. [34], by analyzing the status of cartilage, were able to link the development of osteoarthritis with the availability of lipids. Other more recent studies have reported well-established links with lipid accumulation and the development of OA [36, 37], particularly in its early stages before histological changes occur.

In this study, CRM appears to be very useful in differentiating degraded from healthy articular cartilage tissue. Indeed, during the early stages of osteoarthritis, biochemical changes precede the later structural changes and therefore play a fundamental role in contrast to mechanical factors, which are no longer significant in the advanced stages of disease progression. This makes CRM a privileged tool for early detection of ECM degradation. It is now recognized that CRM can be used for quantitative analysis on thicker, unfixed, hydrated, or even submerged samples, as long as water does not interfere with the Raman signals [13]. Moreover, since depth analysis does not require cutting of the sample, it can be applied to healthy tissue, such as articular cartilage.

In any case, this technique seems to be useful in circumstances where tissue biopsies are recorded or even with Raman-compatible arthroscopic probes [38]. As such, numerous Raman spectroscopy devices in the form of fiber optic probes have been developed and tested for some on human knee joint tissue [39, 40], a common anatomical site for arthroscopic surgery, and allowed detection of cartilage with contributions from subchondral bone [41], others on the colon for real-time *in vivo* assessment of adenomatous polyps [39], or either to nondestructively monitor the

growth of *in vitro* tissue engineering constructs in real time for regenerative medicine applications toward controlled clinical translation [38, 40]. As pointed out by Karen et al. [41], fiber optic probes are a convenient format to couple optical spectroscopies to an arthroscopic probe because they are compatible with the small dimensions of the arthroscope and provide the same chemical information available in a microscopy instrument. In this sense, we agree with Gao et al. [42] that CRM has the potential to be incorporated into a fiber-optic probe device to build a fiber-optic-based confocal Raman microscopy detection unit for an arthroscope for clinical use. As a result, it has the capabilities during arthroscopic procedures to remove the operator from damage to healthy tissue that is usually, in addition to degraded cartilage or sclerotic bone, sometimes included at the microfracture site [41]. Therefore, the coupling of confocal Raman microscopy to arthroscopy could facilitate not only to specifically identify degraded or damaged tissues, but also to better guide surgical interventions by avoiding surrounding healthy tissues. Obviously, such a technology practiced on a daily basis in the clinical setting would strongly contribute to prevent irreversible cartilage damage in the more advanced stages and would favor the institution of adequate management either by stem cell injection to induce regeneration of damaged sites or by other therapeutics without impacting surrounding healthy tissue.

#### **6. Conclusion**

Osteoarthritis is a degenerative disease that primarily affects articular cartilage and related joint tissues and imposes an increasing social burden due to overall activity limitation, especially in the elderly. With degradation, the major components of the cartilage ECM, particularly collagen and proteoglycans, are progressively degraded by the released inflammatory factors. Currently, the diagnosis of OA relies on radiographic methods based on the Kellgren-Lawrence scores, in which joint space narrowing is considered the main diagnostic indicator of advanced disease. One of the major challenges in the management of OA is the ability to make an early diagnosis. To date, there are no biomarkers available for early diagnosis of the disease and no effective therapy other than symptomatic treatment and joint replacement surgery.

The results of our study demonstrate the ability of CRM to differentiate damaged from healthy articular cartilage tissue and thus introduce RCM as a future diagnostic tool for the efficient management of OA. The application of CRM has proven to be relevant in providing biochemical information needed to characterize OA cartilage. Combined with multivariate analysis, CRM is able to identify biomarkers to characterize biochemical and structural changes in articular cartilage ECM. The peaks at 864, 929, 945, 1107, 1271, 1386, and 2887 cm−1 identified in this work can be considered as major indicators to monitor the physio-pathogenesis of articular cartilage. Since *in vivo* Raman spectra have been reported to be collected from human skin, lung, and bone, this technique can therefore advance the diagnostics of AO at an early stage.

#### **Acknowledgements**

N'DRE NJ acknowledges the financial support of the scholarship program of the Ministry of Higher Education and Scientific Research of Côte d'Ivoire. We also thank *Characterization of Degraded Cartilage Using Confocal Raman Microscopy DOI: http://dx.doi.org/10.5772/intechopen.107310*

Dr. Friedrich Menges, who kindly made available his Spectragryph® software for the analysis of our spectral data.

#### **Authors Contributions**

N'DRE NJ was responsible for the realization of the experiments and the preparation of the manuscript. H. SALEHI supervised the Raman experiments and data analysis. M. MAUMUS prepared the samples for the Raman measurements, and ANURADHA R helped us with the analysis and interpretation of the PCA data. D. NOËL supervised the sample preparation and data analysis. KOFFI-GNAGNE and F. CUISINIER edited the manuscript. All authors contributed to the manuscript with comments and suggestions before publication.

#### **Conflicts of interest**

No conflicts of interest are reported.

#### **Author details**

N'Dre Jean1,2\*, Hamideh Salehi1 , Marie Maumus3 , Danièle Noël3 , Yolande Koffi-Gnagne2 and Frédéric Cuisinier1

1 Bioengineering and Nanosciences Laboratory UM\_104, University of Montpellier, France

2 Faculty of Odontostomatology of Abidjan, University Félix Houphouët BOIGNY, Ivory Coast

3 IRMB, University of Montpellier, CHU of Montpellier, France

\*Address all correspondence to<EMAIL_ADDRESS>
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2

## Cartilage Regeneration and Biologics

#### **Chapter 3**

## Evolution of Cartilage Repair Technology

*Chao Zhou, Qi Heng Chen, Zhanzhen Li, Xin Wang, Jiang Peng and Changlong Yu*

#### **Abstract**

Articular cartilage plays an important role in daily joint activities. With the aging of the social population, the degenerative cartilage injury and the sports injury caused by inappropriate exercise of young patients, etc., the incidence rate of articular cartilage injury is constantly rising, and the injured patients tend to be younger. Although articular cartilage has its corresponding metabolic activities, it is difficult to recover and regenerate itself once it is damaged due to lack of nerve, blood vessel, and lymphatic tissue Common articular cartilage injuries can be divided into three types according to the degree of injury: partial cartilage injury, full-thickness cartilage injury, and osteochondral defect. If partial cartilage damage and full-thickness cartilage damage are not found and treated in time in the early stage, further deterioration will lead to serious osteochondral defects. After the corresponding subchondral bone injury, the upward invasion of the upper cartilage layer will also cause the overall osteochondral injury. Therefore, whether the osteochondral injury caused by the topdown or the osteochondral injury caused by the bottom-up, it seriously affects the normal activities of human joints. It not only brings great inconvenience to the daily life of patients, but also causes huge economic and psychological burden to patients. At the same time, it also consumes a large number of social public medical resources. Therefore, seeking an effective osteochondral repair strategy is not only the urgent need and hope of the society, but also one of the clinical scientific problems that clinicians and scientists urgently need to solve.

**Keywords:** articular cartilage, osteochondral defect, scaffold, autologous chondrocyte transplantation technology, cartilage repair

#### **1. Introduction**

The microfracture technique creates a channel between the cartilage defect and the underlying bone marrow by opening the subchondral bone [1]. At present, it is generally believed in clinical research that pluripotent bone marrow mesenchymal stem cells are released and recruited to the defect site through these channels to repair articular cartilage. This technology is often used in clinic because of its simplicity, rapidity, and low cost. However, this method is only effective for small defects,

and cannot form hyaline articular cartilage after repair. It can only provide relative functional improvement after operation, and its clinical effect and significance are relatively limited.

#### **2. Osteochondral transplantation**

Autogenous osteochondral transplantation is also used in clinical practice. It is to take out cylindrical osteochondral tissue from the cartilage surface of the non-load bearing area of the joint and implant it into the cartilage defect of the load-bearing area. Although it has been reported in the literature that good clinical results can be obtained by autologous bone and cartilage transplantation, the results vary greatly depending on age, sex, and lesion size. At the same time, the injury and discomfort of the donor site and the limited availability of local donor tissue make the autologous bone and cartilage transplantation only suitable for some small- and medium-sized cartilage defects. At the same time, there are problems in the repair and healing of cartilage between the transplanted bone and cartilage, and the healing of the transplanted cartilage and the surrounding cartilage of the recipient area. In addition, the adverse effects of the wound opening caused by the injury of the donor area and the release of bleeding inflammatory factors on the microenvironment and homeostasis of the joint make its application limited and gradually reduced. Although allogeneic osteochondral transplantation does not have the problems of donor damage and insufficient graft size, there are problems such as the preservation of allografts, the availability of tissues, the immune response of recipients, and the shortage of donor sources and quality that are also practical problems in clinical application.

#### **3. Scaffold**

In recent years, with the emergence and continuous development of tissue engineering regenerative medicine, it has brought new hope for the repair and regeneration of bone and cartilage after injury. Tissue engineering regenerative medicine mainly includes three factors: biological scaffolds, seed cells, and growth factors [2]. The scaffold of cartilage tissue engineering technology is equivalent to the extracellular matrix. It should be non-toxic, not causing inflammation, and has high porosity. It can provide a good microenvironment for cell growth and can still maintain its shape after implantation [3]. The earliest scaffold materials used in cartilage tissue engineering are PGA, PLA, PLGA, etc. [4]. When PRP gel is used as a scaffold alone, it is easy to squeeze and deform after subcutaneous implantation, and specific cartilage tissue cannot be formed [5, 6]. 3D printing technology can be used to prepare highprecision scaffolds [7, 8], Plga/dacecm tissue-engineered cartilage scaffolds were prepared by low-temperature deposition 3D printing technology [9, 10], and the results showed that the scaffolds were not cytotoxic and has excellent performance [11]. Lin's [12] study also confirmed that this scaffold can better promote the proliferation of bone marrow mesenchymal stem cells and promote the formation of new cartilage. By integrating the preparation process, it will be a new research direction to construct tissue engineering cartilage scaffolds that cannot only recruit endogenous stem cells but also facilitate the maturation of new tissues.

**The first generation of autologous chondrocyte transplantation technology** is to take articular cartilage under arthroscopy, isolate chondrocytes, culture and

#### *Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

expand them *in vitro*, inject cell suspension into the defect, and finally cover with autologous periosteum for suture. This method can repair cartilage damage with a depth of more than 6–8 mm [13]. However, there are also many disadvantages, such as the leakage of cell suspension and the proliferation of periosteum, which require arthroscopic surgical resection [14].

**The second generation of autologous chondrocyte transplantation technology** is to use some biofilms, such as collagen membrane, to suture with surrounding tissues instead of periosteum. Although the cost is higher than that of autologous periosteum, it does not require secondary surgery, so it is more economical [15].

**The third-generation matrix-induced autologous chondrocyte transplantation** is to expand the chondrocytes cultured *in vitro*, implant them into the rough surface of the I/III double-layer collagen membrane, and then replant them. Finally, the more biocompatible fibrin glue is used for adhesion, eliminating the need for suturing, making the operation easier and reducing the risk of cell leakage [16].

**The fourth-generation cartilage repair technology** With the continuous development of cartilage repair technology, after the iterative upgrading of the previous three generations of cartilage repair technology, cartilage repair technology has reached a new height. Although the third-generation cartilage repair technology overcomes the shortcomings of the previous two generations of repair technology, such as poor survival of cells in the defect area, poor long-term repair effect, uneven articular surface, and so on, some problems in the past have not been solved. For example, articular cartilage is composed of hyaline cartilage, which is still replaced by fibrocartilage after repair, resulting in unsatisfactory mechanical properties and biological properties.

How can we repair cartilage damage and solve these problems at the same time? With the further in-depth study of the cell microenvironment, it is found that the principle of microfracture technology, for example, is to introduce stem cells in bone marrow into the cartilage defect area, so that the stem cells can grow, and differentiate and repair in the microenvironment of the cartilage itself, playing a good repair effect. It is the so-called "one side of the soil nourishes one side of the people." The scaffold we made not only carries the cell load, but also wants it to grow and differentiate well on the scaffold, which has been imitating the internal environment of the machine. So why not use the microenvironment of the cell itself to make this cell carrier? Therefore, the concept of extracellular matrix (ECM) came into being. It can not only provide a good microenvironment for cell growth and differentiation, but can also solve the problems of cell metabolism and biomechanical properties.

#### **3.1 Extracellular matrix (ECM)**

Extracellular matrix (ECM) is a matrix structure that is synthesized and secreted by cells and distributed on the cell surface or between cells. It is a complex network composed of proteins and proteoglycans, which can provide support for tissues and regulate cell functions. It is the dynamic microenvironment of the stem cell niche. Therefore, it has attracted the continuous attention of tissue engineering researchers [17, 18]. In addition, cartilage extracellular matrix can activate intracellular signal transduction pathways through various growth factors and cytokines [19]. Due to the complex composition of extracellular matrix, it is almost impossible to fully bionize the cartilage extracellular matrix in terms of composition, morphology, and function in the current progress of cartilage tissue engineering. At present, the bionic biological scaffold materials used in cartilage tissue engineering include extracellular

matrix-derived materials and non-extracellular matrix-derived materials. The extracellular matrix-derived materials include simple-component materials extracted from the extracellular matrix, mixed-component materials, and tissue acellular extracellular matrix materials. The extracellular matrix-derived material is closer to the cartilage extracellular matrix than other materials in composition and is a very excellent biomimetic scaffold material.

materials include collagen, glycosaminoglycan, hyaluronic acid, gelatin, chondroitin sulfate, tissue acellular extracellular matrix, etc. These materials can be used alone to make scaffolds, but it is often difficult to imitate all components of the cartilage when applied alone. Therefore, some researchers have also combined two or more of these materials, or combined with other natural materials or artificial materials [30, 31]. Extracellular matrix-derived scaffolds can be divided into four categories according to their composition: monomeric natural polymer materials, multiple natural polymer mixed materials, new biomaterials constructed by combining natural polymer materials with synthetic polymer materials, and tissue acellular extracellular matrix materials.

3.Preparation of tissue acellular extracellular matrix scaffold: Cartilage tissue needs to go through two very important steps, tissue acellularization and scaffold preparation, before it can be prepared as a scaffold derived from extracellular matrix. The purpose of tissue decellularization is to remove the substances that cause immune reaction, such as cell membrane materials, soluble proteins, nucleic acids, while retaining the extracellular matrix components of cartilage as much as possible and maintaining its biological activity. At present, there are two kinds of commonly used decellularization methods: physical method and chemical method. The physical methods include freezing and thawing method, mechanical oscillation method, differential centrifugal method, etc. Chemical methods include enzymatic digestion, high or low osmotic solution decellularization, acid-base decellularization, etc. Usually, one method or a combination of methods is used in the process of decellularization. When preparing acellular extracellular matrix materials, more attention should be paid to whether acellularization is complete and the loss of components. When the extracellular matrix material is prepared, it needs to be made into a scaffold. At present, many methods have been developed to prepare cartilage tissue engineering scaffolds. Different preparation processes can have obvious effects on the performance of scaffolds. In practical application, different preparation processes can be selected according to the conditions [29]. Freeze drying method: The extracellular matrix slurry is poured into the mold, freeze-dried at low temperature, then crosslinked, and finally sterilized to form a 3D scaffold. Immunogenicity and toxicity are removed. Cartilage acellular extracellular matrix scaffold material has many advantages. It is completely derived from biological tissue. After acellular treatment, the immunogenicity is removed to the maximum extent, and many effective components of natural cartilage extracellular matrix are retained. It can provide a better microenvironment for seed cell growth than pure component materials and artificial mixed materials. The extracellular matrix contains natural cytokines, which can promote the proliferation and differentiation of seed cells without adding exogenous cytokines. The PLA General Hospital adopted its own unique preparation process to form a biphasic-oriented stent. The cells implanted in the scaffolds were induced to differentiate into chondrocytes and grow in the direction induced by the scaffolds. Finally, the regeneration and growth of the repaired cartilage become similar to the natural cartilage.

The prepared scaffold had three-dimensional porous sponge-like longitudinally oriented structure. There were cartilage fibers around the scaffold pores. After hematoxylin-eosin staining, no nucleus was observed. Both safranin O and Sirius red staining confirmed that cartilage tissue engineering scaffold contained collagen and

cartilage matrix. The porosity and water absorption of the scaffold was (91.8 ± 2.9)% and (93.5 ± 1.4)%, respectively. MTT results showed that the leaching liquor of human cartilage-derived extracellular matrix was non-toxic to chondrocytes. After co-culture, human chondrocytes adhered, proliferated, and evenly distributed on the peripheral wall of the scaffold pores. The results showed that human articular cartilage-derived extracellular matrix had similar composition to natural cartilage, provided the structure suitable for cell adhesion and proliferation, and exhibited good histocompatibility. Therefore, human articular cartilage-derived extracellular matrix can be used as a scaffold material for repairing cartilage defects by tissue engineering technique.

*ECM scaffold preparation, composite cells, cartilage defect model, and treatment plan [33].*

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

> cartilage tissue components, has good proliferation and differentiation promoting effects on seed cells, has biomechanical characteristics close to cartilage, and can be degraded *in vivo* and will not cause adverse reactions. Extracellular matrixderived scaffolds have been used in cartilage tissue engineering and clinical practice because of some characteristics of biomimetic scaffolds, and have been proved to be good biomimetic scaffolds for cartilage tissue engineering. However, the shortcoming is that the scaffold materials derived from extracellular matrix have poor mechanical properties and are not easy to be processed. Extracellular matrix as a scaffold material also has its disadvantages. Because there is almost no way to maintain the original physical properties of cartilage tissue during the fabrication process, the fabricated extracellular matrix scaffold cannot reach the level of natural cartilage in terms of biomechanical properties, and atrophy occurs after seed cells are planted. However, these problems can be greatly improved by using cross-linking method during the fabrication of scaffold. Rowland et al. [32] analyzed the effects of heat crosslinking treatment, ultraviolet irradiation, and chemical crosslinking agent carbodiimide on the contraction of scaffolds, combined with adult bone marrow-derived stem cells for chondrocyte differentiation culture, and proved that crosslinking (non-crosslinking as control) can prevent cell-mediated contraction of extracellular matrix scaffolds (**Figures 1**–**4**).

Therefore, the future research direction must focus on making full use of the existing materials, continuously improving the preparation process, combining synthetic materials with extracellular matrix-derived scaffold materials to prepare

**Figure 2.**

*ECM scaffolds after decellularization of cartilage tissue, and then recombined with autologous chondrocytes or adipose stem cells [33].*

#### **Figure 3.**

*The ECM scaffolds of cartilage tissue after decellularization treatment were then compounded with autologous chondrocytes or adipose stem cells ECM scaffolds. After the cells were compounded, cell viability and toxicity tests showed that the cells survived well and had strong activity on the scaffolds [33].*

cartilage tissue engineering scaffolds, and further exploring methods to change the properties of various materials. That is, the fifth-generation scaffold micro-tissue composite biomimetic scaffold mentioned later (**Figures 5**–**7**).

Regeneration of articular cartilage is one of the most serious problems facing joint surgeons. In recent years, microcarrier applications have made great progress in cartilage tissue engineering. One advance is the cost-effective expansion of seed cells that provide the necessary microenvironment for cells. Furthermore, microcarriers can also carry proteins, factors that are beneficial for cartilage repair and drugs for cartilage regeneration. Some microcarriers have the advantages on injection. The use of microcarriers having these features avoids the disadvantages of conventional methods and provides unique advantages. For clinical transformation potential, microcarriers have many advantages, such as supplying plenty useful cells, factors, drugs,

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

#### **Figure 4.**

*The ECM scaffolds after decellularization of cartilage tissue, and then composite with autologous chondrocytes or adipose stem cell ECM scaffolds and composite cells to repair the cartilage defect of femoral condyle in rats [33].*

microenvironment. Microcarriers also have many application features. First, they can be injected directly into the corresponding site to weaken invasiveness. Second, they can be implanted after organoid formation to enhance repair efficiency. Finally, combining with scaffolds can meliorate the mechanical deficiencies of microcarriers. Thence, the application of microcarriers has great potentiality for clinical translation. A brand new application of microcarriers on tissue engineering is to place them inside hydrogels to make scaffolds or bioinks. Tissue engineering may revolutionize the status quo of cartilage regeneration. However, achieving clinical translation still requires a lot of research support.

#### **Figure 5.**

*Pathological section of cartilage defect repaired by ECM scaffolds after decellularization of cartilage tissue and then compounded with autologous chondrocytes or adipose stem cell ECM scaffolds [33].*

#### **Figure 6.**

*Pathological section of cartilage defect repaired by ECM scaffolds after decellularization of cartilage tissue and then compounded with autologous chondrocytes or adipose stem cell ECM scaffolds [33].*

The advantages of microcarriers are discussed by comparing the characteristics of the microcarrier with other traditional methods. We also discuss the utilization potentiality of the microcarrier and the prospect of future development.

Articular cartilage is very important in the human body. Due to the avascular nature of articular cartilage, it is basically unable to achieve self-healing. Therefore, the treatment of articular cartilage damage is a serious problem for orthopedic

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

**Figure 7.**

*MRI image of cartilage defect repaired by ECM scaffolds after decellularization of cartilage tissue and then compounded with autologous chondrocytes or adipose stem cell ECM scaffolds [33].*

surgeons. If not treated properly, articular cartilage defects can easily lead to osteoarthritis or accelerate the progression of osteoarthritis (OA). OA is one of the most common degenerative joint diseases, and its most notable features are pain and limited joint mobility. The most commonly used methods for clinical treatment of cartilage injury today include microfracture and surgical lavage. Traditional cartilage repair techniques in clinical treatment include bone marrow stimulation techniques (BMS), synthetic, etc. However, all these traditional techniques have shortcomings, such as the inability to repair large-scale cartilage damages, the high rate of secondary operations, and the unsatisfactory prognosis. Tissue engineering (TE) technology has developed speedily in recent decades. Accordingly, a new method for cartilage defect repair is provided. However, traditional tissue engineering techniques have three drawbacks in cartilage regeneration. First, chondrocyte loses certain phenotype in *in vitro* expansion. Second, traditional three-dimensional porous scaffold has hollow phenomenon. Third, tissue-engineered cartilage takes a long period of time to repair the cartilage defect in *in vivo* models. In the field of tissue engineering for cartilage regeneration, many progress have been made in the study of cartilage repair using microcarriers. A microcarrier is a microparticle that can carry cells, factors, or drugs, with a diameter of about 100–300 microns [34]. Microcarriers provide some new ideas for clinical treatment of cartilage injury-induced OA.

#### **4. Traditional methods for treating cartilage injury and characteristics of microcarrier methods**

The function and quality of articular cartilage deteriorates with age. Cartilage damage usually progresses from the surface of the articular cartilage to the subchondral bone, leading to the generation of OA.OA creates a huge economic and social load. Degenerative joint damage is often accompanied by cartilage loss. The study reported that cartilage defects in OA patients with symptoms for more than 2 years were more likely to have accelerated progression.

Hence, the treatment of articular cartilage damages is especially important. The traditional techniques commonly used are as follows: first, BMS technology; second, filling cartilage damages with biological tissue; third, cartilage cell implantation; fourth, use metal or other artificial materials to repair cartilage defects; fifth, cell therapy; sixth, drugs that stimulate cartilage repair.

Microfracture technique is a common technique for repairing cartilage damage in BMS technology. It repairs bone marrow defects by transferring bone marrow mesenchymal stem cells (BMSCs) from the intramedullary cavity to the surface of the cartilage damage through fracture drilling. However, the chondrogenic potential of BMSCs is significantly reduced in the elderly [35]. If the cell source is coupled with the disease state, the repair effect is greatly diminished. Therefore, "vivacious" BMSCs are at the heart of the microfracture technique approach for cartilage repair. The use of autologous cartilage as a biological tissue to fill cartilage defects is also a representative method to repair cartilage damage [35]. Osteochondral transplantation can effectively treat knee joint cartilage friction injury. Symptoms of successful transplant patients improved significantly, but this method does not guarantee a good prognosis and a high success rate. However, not only does the surgery have a low success rate, but autologous cartilage transplantation itself is a method of repairing damaged cartilage by destroying healthy cartilage. Fresh allogeneic osteochondral grafts are primarily used to treat young patients with extremely severe articular cartilage damage. Although allogeneic cartilage transplantation has been reported to relieve symptoms of cartilage damage, the scarcity of donor sources has made it impossible to expand the scope of this technique.

Finding a viable treatment to repair damaged cartilage before osteoarthritis develops has become a worldwide problem. Cartilage TE technology emerges as a new proven treatment that offers hope to those who need to treat articular cartilage injuries. Cartilage TE uses cell differentiation and proliferation factors to culture and expand cartilage seed cells *in vitro*, co-culture high-quality seed cells with bioscaffold materials, fill them into the damaged area of cartilage, and gradually combine with the original cartilage to form new cartilage tissue.

Microcarriers [34] are a type of small functional particles with a diameter of about 100–300 μm. Microcarriers contain a wide variety of materials with good biocompatibility, which can boost seed cells as a suitable support matrix. Compared with the traditional methods, functional TE microcarriers can repair cartilage damage, which can not only play the unique advantages of microcarriers, but also avoid some disadvantages of traditional methods.

Advantages of microcarriers over conventional techniques: First, an important advantage of microcarriers is that they can be loaded with sufficient seed cells, which expand *in vitro*. More BMSCs are carried on microcarriers than BMSCs released from BMS. If microcarriers are fabricated by electrospray method, a large amount of BMSCs can theoretically be carried [36]. Second, the microcarriers made of high-quality biomaterials also take full advantage of the traditional method of filling cartilage defects with biomaterials. Second, microcarriers can also be used to treat cartilage damage by using traditional methods of filling cartilage defects with biomaterials. For example, alginate-based microcarriers resemble extracellular matrix (ECM) and can promote cartilage regeneration even without seed cells. Third, in a sense, microcarriers also belong to an artificial material. High-quality microcarriers with seed cells are cultured to constitute tissue-engineered cartilage microtissues. Microtissue [37] provides cartilage tissue to damaged areas of cartilage in a manner similar to cartilage grafting. The application of microcarriers has

opened up a new way for the treatment of cartilage injury by the method of artificial material implantation. Fourth, microcarriers can target delivery of drugs such as chondroitin sulfate and glucosamine sulfate that are beneficial for the treatment of cartilage damage.

#### **5. Summary of the advantages of microcarriers in the treatment of articular cartilage injury**

TE aims to develop biological products to replace damaged tissues or organs with the goal of restoring normal function. Current TE therapies for cartilage regeneration are dominated by the use of synthetic or natural implants. A sufficient amount of high-quality seed cells and a proper supporting matrix are the basic conditions for TE. In the field of TE to repair cartilage damage, microcarriers have advantages in every condition. The advantages of microcarriers from these aspects are summarized below.

#### **5.1 Adequate and "virant" seed cells**

Adequate and high-quality seed cells are one of the important components of TE. Microcarriers can carry seed cells and target them to the damaged site for repair. BMSCs have been intensively studied, and many are devoted to their application in cartilage damage repair. BMSCs have been shown to possess immunomodulatory functions, multilineage differentiation potential, and tissue homing properties. Furthermore, it is worth noting that even BMSCs obtained from severe OA patients have chondrogenic capacity and can synthesize cartilage extracellular matrix. Studies [36, 37] pointed out that microcarriers can carry a large number of BMSCs and make them successfully differentiate into chondrocytes, and finally form cartilage TE implants. These studies demonstrate that the microcarriers loaded with BMSCs can be used for cartilage damage repair both *in vitro* and *in vivo*.

Human adipose-derived stem cells (hADSCs) are also able to differentiate into cartilage. A study [38] reported that amplifying ADSCs on microcarriers and then implanting them into defects could well promote cartilage repair. One article [39] demonstrated that rapidly degrading microcarriers promoted cartilage regeneration by promoting the generation of immature bone-like tissue. Another study [40] described the effect of different cell densities and different differentiation states of MSCs in microcarriers on cartilage repair. This research shows that the high density of mesenchymal stem cells is beneficial for cartilage repair, and the well-differentiated state of MSCs also has an important impact on cartilage repair. In another study, the combination of hADSCs and transforming growth factor-beta (TGF-β) 3 microcarriers was much more effective than alone in a rabbit model of OA treatment.

Chondrocytes clearly promote cartilage regeneration [38]. There was no significant difference in yield between chondrocytes carried in dynamic microcarriers and chondrocytes produced in tissue culture plates.

In addition, the seed cells carried by the microcarriers can stay in the cartilage defect for a longer time than the mesenchymal stem cells in the BMS method. One study [37] showed that microcarrier-carried cells or their progeny cells persisted for at least 6 weeks.

#### **5.2 Microenvironment**

The microenvironment is also one of the conditions affecting TE. Microcarriers can provide a favorable microenvironment for cartilage defect repair, such as providing an appropriate matrix to help seed cell adhesion and proliferation or carrying cytokines that promote cartilage formation. The microenvironment provided by the microcarriers also has an impact at the protein level, which can guide seed cells into the cellular matrix, which can then be used to restore damaged cartilage.

The results of genetic analysis showed that the chondrocyte phenotype of chondrocytes expanded by dynamic microcarrier culture did not disappear. In a study of chondrocyte expansion methods using dynamic microcarriers, it was found that it could increase the expression levels of hyaline cartilage proteins such as Col2 and Agg compared with the traditional 2D cultures.

Biomaterials such as gelatin can provide a microenvironment conducive to seed cell proliferation and adhesion. According to research [36], microcarriers containing gelatin can be used to repair cartilage damage because they can promote the proliferation of seed cells.

Biomaterials such as gelatin can provide a microenvironment conducive to seed cell proliferation and adhesion. According to research [36], microcarriers containing gelatin can be used to repair cartilage damage because they can promote the proliferation of seed cells.

ECM proteins also play an important role in promoting cartilage regeneration. Microenvironments maintained by high-quality biomaterials, such as alginate hydrogels, also have the potential to repair cartilage damage due to their properties similar to natural ECM. Biomaterial-based approaches, such as the use of pre-formed hydrogel matrices, can provide mechanical stability while also taking advantage of biochemical incentives to maintain cellular propensity for chondrogenic differentiation. Alginate is very suitable as a raw material for the manufacture of microcarriers. A previous study [41] noted that alginate hydrogel microcarriers provided ECM in chondrocyte culture, promoting cell differentiation toward cartilage, compared to conventional 2D cultures with or without TGF-β1 added. The internal structure of these microcarriers is very similar to that of natural ECM, so they can be used as an alternative ECM for implantation in cartilage defect areas (**Figures 8**–**10**).

The application of appropriate matrix assistance can provide a better microenvironment for cells to differentiate into cartilage. In one study, Ramkumar used microcarriers composed of agarose (AG) and collagen type II (COL-II) to carry bone marrow mesenchymal stem cells. COL-II not only promotes mesenchymal stem cell proliferation but also increases local ECM content [32]; therefore, microcarriers prepared with COL-II and agarose can mimic some of the protein components in ECM to promote cartilage repair. The microspheres prepared by the authors are 80–100 microns in diameter, which does not exactly match the size of the defined microcarriers, but produces basically the same effect. Lineage-specific differentiation of embedded BMSCs did not disappear in culture. More importantly, the microcarriers prepared with COL-II promoted the expression of the chondrogenic phenotype of BMSCs. Furthermore, the microcarriers have excellent physical properties and do not have any negative effects on cell viability. In a study [44], Paulomi Ghosh's group and his team used microcarriers to carry acellular cartilage for related research. The advantage of adding acellular cartilage to microcarriers is that acellular cartilage itself contains a large number of chondrocytes' own proteins, cytokines, GAGs, and TGF,

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

#### **Figure 8.**

*Flowchart of the experimental design of the fresh goat knee cartilage made into pellets through a series of procedures and co-cultured with BMSCs for repairing rat cartilage damage [42].*

#### **Figure 9.**

*Scanning electron microscope observation of cartilage-derived microcarriers Villi-like appearance of cartilage particles [43].*

#### **Figure 10.**

*The cartilage-derived microcarriers stained positively with toluidine blue, and it was strongly positive in the central region near the cartilage particles. The surrounding filament structure is obvious, and it is closely connected with the cartilage particles [43].*

which can provide chondrocytes with a microenvironment that is most in line with the original growth environment of cartilage.

Overall, we can integrate one or more components that are beneficial for the repair of cartilage damage in the microcarrier, provide a microenvironment that is conducive to the growth, proliferation, and differentiation into cartilage of seed cells, and increase the expression of cartilage-related proteins (**Figures 11** and **12**).

#### **6. Suitable supporting matrix**

Appropriate supportive matrix is another extremely important reason why cartilage TE can promote cartilage regeneration. We generally divide microcarriers into two categories according to their sources, synthetic microcarriers and natural source microcarriers. The source of synthetic microcarriers is convenient, but their biocompatibility is poor, almost all lack cell-specific recognition sites, and some have some cytotoxicity. Therefore, researchers are now more optimistic about using natural polymers as materials to prepare microcarriers with good biocompatibility, such as gelatin, alginate, chitosan [41].

One study [37] claimed that chitosan microcarriers combined with crocodile dialdehyde bacterial cellulose and DL-allo-hydroxylysine could promote cell proliferation, growth, and migration. Hydroxylysine is a high-quality amino acid with low immunogenicity and good compatibility. Type II collagen contains a large amount of hydroxylysine, and *in vitro* cell culture, hydroxylysine can also promote cell differentiation into cartilage. In addition, oxidized bacterial cellulose can also be used as a raw material for microcarriers, which not only have structural characteristics similar to natural ECM, but also have good physical properties.

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

In addition, some researchers use both natural and synthetic materials to manufacture the microcarriers. The researchers produced chondrocyte-complexed chitosan polyelectrolyte complex (PEC) microcarriers that significantly outperformed chitosan microcarriers in the ability to generate cartilage matrices. Biomaterials can only be used in the field of cartilage repair if they can promote cell proliferation and differentiation, and have sufficient physical properties. Many scholars have conducted continuous research to make this ideal biological material. Studies by some scholars have shown that porous PLGA microcarriers can promote the formation of cartilage by MSCs. E Filova's team prepared a scaffold of polyε-caprolactone (PCL) porous scaffolds containing chitosan microparticles, which not only possessed good biocompatibility, but also exhibited good mechanical properties. The microcarriers constructed by PCL provide the advantages of sufficiently strong mechanical strength and sufficiently large porosity in cartilage regeneration, and composite chitosan can better promote cartilage repair. And the higher the concentration of chitosan, the better the effect of cartilage repair.

In addition to the above three factors, microcarriers also have many advantages in other aspects (**Figure 13**).

**Figure 13.**

*A is Alcian blue staining of alginate-adipose derived stem cell microspheres after 21 days of chondrogenic induction, blue acid glycosaminoglycan components are visible (scale bar = 100 μm); B is Alcian blue staining of alginate—5 g/L gelatin-adipose derived stem cell microspheres after 21 days of chondrogenic induction, darker acid glycosaminoglycan components were seen (scale bar = 100 μm) [46].*

#### **6.1 Microcarriers assist in the formation of microtissues**

A study [47] showed that the biocompatibility of the scaffolds was better when the scaffolds were first cultured in a perfusion culture system with exogenous stimulation for a certain period of time and then implanted.

During the construction of tissue-engineered cartilage, the ECM secreted by the cells carried by the microcarriers binds each cartilage microtissue or organoid together, thereby promoting the formation of tissue-engineered cartilage. Seed cellbound microcarriers were continuously and dynamically cultured in custom-built bioreactors [37]. When the microcarriers begin to secrete ECM, cartilage microtissues can be obtained. In addition, microtissues have other benefits, because functional microtissues can be implanted into the site of cartilage defects, which avoids damage to cell viability and cell numbers caused by the process of digesting cells before the cells are transferred from traditional 2D cultures.

#### **6.2 Microcarriers are injectable**

Most of today's tissue engineering treatments for articular cartilage defects require opening the joint cavity to implant bioscaffolds, a process that damages the tissue surrounding the joint. Because of its small diameter, microcarriers can be implanted into the cartilage defect site by injection, thereby reducing the damage to the periarticular tissue caused by the surgical incision [48].

However, in terms of the potential of injectable microcarriers to treat cartilage defects, how to retain or adhere to cartilage defects for a long time after implantation of microcarriers is one of the major issues to be solved. Some studies [37] used bioprotein glue to fix the implanted microtissue in the defect. Although there is nothing wrong with the use of biological protein glue, the microtissue needs to be accurately placed in the cartilage defect, which is difficult to achieve in clinical surgery. Several researchers have developed PLGA microtissues coated with magnetic nanoparticles to enable precise localization of cartilage defects by magnetism. The researchers also prepared alginate-based microtissues that

dispensed MSCs and iron oxide nanoparticles (IONPs) separately, preventing potential damage to cells by IONPs. Another advantage of such microspheres is that they can be loaded with a large number of IONPs, which make them easier to move after magnetization. These magnetic microcarriers play an important role in cartilage repair.

#### **6.3 Incorporating bioscaffolds**

Cell-loaded microcarriers can also be used in combination with bioscaffolds for cartilage repair. The combination of PLGA microtissue and collagen/silk fibroin composite scaffold can not only promote cartilage repair, but also promote the fusion of newly formed cartilage with surrounding normal cartilage. It has also been reported that cold atmospheric plasma (CAP)-modified electrospinning scaffolds combined with microtissues can improve the proliferation and differentiation of seeded cells. Another study added hyaluronic acid to the collagen/silk fibroin scaffold, giving it new advantages such as anti-inflammatory and analgesic. At the same time, the researchers added velvet antler polypeptides (PAPs), which can promote cartilage healing, to PLGA microcarriers without seed cells, and found that the scaffolds also promoted the repair of damaged articular cartilage.

#### **6.4 Drug delivery with microcarriers**

Microtissues can also be used for drug delivery. Some researchers have repaired pig cartilage by using microtissues to deliver BMP-2 and TGF-3 at the site of injury and release them continuously. One study [49] demonstrated that microtissues containing chondroitin sulfate, a drug that favors cartilage repair, were superior to microfracture techniques for cartilage regeneration. The use of microcarriers as an injectable drug delivery system is not only feasible, but also simple and easy to implement, and effective in the treatment of cartilage damage [50, 51] .The authors [50] infiltrated PLGA microtissues in fluvastatin, which had a significant effect on reducing cartilage degeneration in rabbits. They [51] added tumor necrosis factor-α-stimulated gene 6 in PLGA microtissues and achieved obvious effects in repairing cartilage defects in rats.

In conclusion, microcarriers have multiple and distinct advantages in cartilage repair.

#### **7. Prospects Application prospect of microcarriers in the treatment of cartilage injury**

The ability of microcarriers to provide a suitable microenvironment is very important for the repair of cartilage defects. In addition, some studies [37] have shown that microcarriers whose structures are more similar to the cartilage tissue's native ECM perform better in repairing articular cartilage damage. In addition, other studies [48] have shown that there is a higher rate of cell attachment if the cartilage tissue microcarriers contain ECM. Therefore, we reasoned that if the native ECM could be directly incorporated or generated in microcarriers with appropriate physical characteristics, the effect of cartilage repair would be improved.

It is also an unsolved problem at this stage to find raw materials for the production of microcarriers that can perfectly replace the natural cartilage ECM. At present,

there is also a lack of research on whether microcarriers prepared by composite materials can effectively promote the growth, proliferation, and differentiation of seed cells.

Physical properties such as porosity and volume have become key factors affecting the efficacy of cartilage repair. The future development direction of microcarriers should be how to determine the appropriate porosity and volume according to the specific conditions of patients, and establish an efficient way to calculate these physical properties, which will play an important role in future clinical applications.

The source of cells is also an important condition that affects the application effect of microcarriers. The source of chondrocytes is often the patient's own, and obtaining chondrocytes will damage the cartilage. In contrast, ADSCs may be a better choice. Another point to study is how much the patient's age, disease, etc., affect the quality of the obtained seed cells.

Injectable microcarriers allow the treatment of cartilage injuries to be performed minimally invasively, reducing damage to surrounding tissue from the incision. The research and development of magnetic microcarriers has greatly solved the problem that it is difficult to accurately locate cartilage defects by injection methods [37]. If we continue to conduct more in-depth research in this area, or find other methods that can accurately locate under injection treatment conditions, the treatment of cartilage injury with microcarriers is one step closer to clinical application.

Although many *in vivo* and *in vitro* experiments have demonstrated that microcarriers can repair cartilage well, they have not yet reached the clinical trial stage. Orthopedic surgeons who want to pay attention to OA treatment can continue to pay attention to the research progress of microcarriers and confirm their ability to repair human cartilage through clinical studies.

#### **Author details**

Chao Zhou1 \*, Qi Heng Chen2 , Zhanzhen Li1 , Xin Wang2 , Jiang Peng2 and Changlong Yu2

1 Zhoushan Dinghai Guanghua Hospital, Zhoushan, China

2 Institute of Orthopaedics, Beijing Key Lab of Regenerative Medicine in Orthopaedics, Chinese PLA General Hospital, Beijing, China

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Evolution of Cartilage Repair Technology DOI: http://dx.doi.org/10.5772/intechopen.108031*

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#### **Chapter 4**

## Bio-Orthopedics: A New Approach to Osteoarthritis and Joint Disorders

*Alberto Gobbi, Katarzyna Herman and Dawid Szwedowski*

#### **Abstract**

Osteoarthritis is a major cause of functional limitation and a raising burden in aging population. Lately more research is directed into finding biological enhancement of healing processes in joint dysfunctions. Biological cell-based therapies for cartilage restoration treatment were created to address the need for the long-term viability of repaired tissues. Additionally, the use of biologic therapies is also considered in common disorders affecting ligaments and cartilage. However, if inevitable arthritic changes commence biological therapies offer options to delay the need for arthroplasty. This chapter provides insights into these regenerative, joint preservation techniques for cartilage treatment, osteoarthritis, and other joint disorders.

**Keywords:** cartilage, osteoarthritis, bio-orthopedics, PRP, BMAC, knee articular preservation

#### **1. Introduction**

Osteoarthritis (OA) is one of the most common joint diseases; characterized mainly by joint pain and functional impairment, due to cartilage degeneration, subchondral bone remodeling, and synovial inflammation [1]. OA affects 7% of the global population [2], making it an important problem to solve for the orthopedic surgeons.

Tissue healing is limited not only by the biochemical environment but also by other coexisting factors such as diabetes, smoking, hypercholesterolemia or local factors, which can further weaken healing [3]. Biological response has been studied for many years in order to optimize the healing processes. These have resulted in cellbased, cytokine-based, and scaffold-based therapies [4]. In this chapter, we analyze current concepts in the use of biological treatments. This book chapter intends to provide a review of the current status of biological therapies for OA and other joint disorders in orthopedics.

#### **2. Biological options**

#### **2.1 PRP**

Lately, research in OA has moved the attention toward biochemical pathways that can be aimed therapeutically through biological intervention. In the past decade, a great interest has been focused on platelet-rich plasma (PRP). It is one of the "hot topics" of regenerative medicine due to its potential to help in different conditions. PRP has surfaced as a biological therapy for the treatment of cartilage injuries and for intraarticular application to address knee pain. PRP contains cytokines, growth factors, and inflammatory mediators, all capable of stimulating cartilage, subchondral bone, and soft tissue healing [5]. PRP contains a significant number of growth factors and proteins in the alpha granules of platelets, that were observed to have regenerative and analgesic effect [6, 7]. What is more, studies demonstrated that platelet-rich plasma (PRP) has an anti-inflammatory influence and counteracts catabolic processes within the joint [8–10]. Additionally, it has been shown to promote chondrocyte proliferation and increase the synthesis of collagen and proteoglycans and for this reason, the application of PRP for osteochondral pathologies has increased [5, 11]. PRP can be categorized as leucocyte-rich PRP (L-PRP), leucocyte-poor PRP (LP-PRP), or platelet-poor plasma (PPP) depending on the preparation technique. The ingredients of PRP categories vary, depending on which system is used to prepare the autologous blood, so care must be taken when choosing the method. There is no one gold standard, so injections of PRP can be done once or in cycles of 3 or more injections. PRP is acquired from patients' peripheral blood. The venous blood is centrifuged in a special probe, according to instructions provided by the manufacturer. When using intra-articularly the PRP is injected into the joint after careful skin disinfection at the needle entry point. The highest beneficial effect is seen at 6 months after cycle of injections, however it may last up to two years [12]. Authors of a meta-analysis of randomized controlled trials using PRP in treatment of knee OA concluded that a statistically significant beneficial effect over placebo was observed at 6 and 12 months [13]. It was also shown that PRP is more effective both in short- or long-term pain and functional recovery when compared to HA [14].

#### **2.2 Hyaluronic acid**

HA is a non-sulphated glycosaminoglycan that consists of D-glucuronic acid and N-acetylglucosamine units. It ensures tissue hydration due to its hydrophilic properties and high solubility in an aqueous environment [15]. In a healthy joint it is produced by the synovium, exactly the type B synoviocytes and fibroblast. Its role is to preserve the viscoelastic and functional characteristics of the articular cartilage [16] in the same time promoting chondrocytes proliferation and differentiation [17]. Additionally, it provides joint lubrication and shock absorption. Interestingly in a study HA was shown to inhibit tissue nociceptors [16]. HA also inhibits IL-1β-induced oxidative stress and the inflammatory mediators, such as metalloproteases (MMP-13), nitric oxide (NO), and prostaglandin (PGE2) [18]. Various HA compounds are available that differ in molecular weight (MW), composition, and dosage regimens. High MW HA consists of molecules ranging from 3000kDa to 6000kDa; medium MW from1500 to 3000kDa; low MW has been described as ≤1500kDa. High molecular weight was proven to increase the fluid retention into the joint and to have greater anti-inflammatory effect than low MW HA [19]. Clearance rate of HA is another important factor influencing its effectiveness. That is why stabilizers have been

*Bio-Orthopedics: A New Approach to Osteoarthritis and Joint Disorders DOI: http://dx.doi.org/10.5772/intechopen.110845*

introduced to slow down the clearance rate. One of the used options is trehalose, a disaccharide that acts as a protector of HA. In an in vitro model hyaluronic acid combined with trehalose had improved resistance to hyaluronidase enzyme compared to standard therapy [20]. Additionally in a randomized controlled trial comparing 1% trehalose hyaluronic acid (T-HA) versus non-trehalose hyaluronic acid (NT-HA) for patients with OA KOOS, VAS and IKDC improved for T-HA group at 6 months post-injection while for NT-HA patients it decreased to baseline. The difference was reported statistically significant (*P* < 0.05) [21]. At our institution we are currently conducting two clinical trials on the use of new formulations of HA for the treatment of knee OA. Particularly, one consists of a high chain (1800–2600 KDalton) combined with Niacinamide, an excipient that protects the acid from hyaluronidase degradation activity, promoting longer HA stability. Our preliminary results confirm a more durable effect of HA with Niacinamide compared to standard HA in patients affected with knee OA, at 6-month follow-up. We are also evaluating the efficacy of a second product of HA combined with Collagen type I which reinforces the cartilaginous matrix deteriorated by the ongoing pathological processes of osteoarthritis. We are testing it in young athletic patients to evaluate if they can have more benefits when compared with other therapies.

#### **2.3 Adipose stem cells**

An Autologous Microfragmented Adipose Tissue (MFAT) and Stromal Vascular Fraction (SVF) in contrast to peripheral blood has 25,000 times more reparative cells [22]. MFAT is acquired from adipose tissue from abdominal or supragluteal area with a special lipoaspirate cannula. Lipoaspirate is transferred to the special low-pressure cylindrical system and combined with saline solution and then mixed. A final product is a concentration of pericytes and MScs. This is then applied into the joint through an injection or during arthroscopy. Promising results have been shown in literature. In multi-centric, international, open-label study evaluating the outcomes of MFAT injections in patients with knee OA at 2-year follow-up significant functional and quality of life improvement was seen in 88% of patients [23]. In a prospective randomized control trial comparing leukocyte-poor PRP combined with HA and MFAT no significant superiority was seen for either group, both procedures were found to be safe with no major complications showing good results at mid-term follow-up [24]. Additionally, a new technique using SVF and platelet-rich plasma to treat knee osteoarthritis (OA) has been recently described. First, the adipose tissue is harvested from abdominal area with the harvester cannula connected to syringe (**Figure 1**). Meanwhile, leucocyte-poor platelet-rich plasma (LP-PRP) preparation is performed. The harvested fat is transferred to an ACP double syringe for first-round centrifugation with an end product of a middle fraction that is the condensed fatty tissue containing the stromal vascular fraction (SVF) (**Figure 2**). After fragmentation the lipoaspirate is transferred back to the ACP double syringe for second-round centrifugation. After centrifugation the SVF fraction is retrieved. Lastly the SVF is combined with LP-PRP injected into the patient's knee joint (**Figure 3**) [25].

#### **2.4 Bone marrow aspirate**

MSCs have multi-differentiation ability, which is why they have been identified as an attractive cell source to regenerate tissue. MSC represents only 0.0001 to 0.01%

#### **Figure 1.**

*Lipoaspiration with a 2.1 mm × 15 cm harvester cannula connected to syringe with Johnnie snap locking system through 5 mm skin cut after infiltrating the fatty tissue with a tumescent solution.*

mononuclear cells in bone marrow aspirates [26]. Bone marrow aspirate concentrate (BMAC) consists of variety of growth factors such as the platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), and bone morphogenetic proteins (BMP)-2 and BMP-7, known to have anabolic and anti-inflammatory effects [27]. Hypothetically MSCs, rather than contributing to tissue formation, act as siteregulated "drugs stores" by releasing immunomodulatory factors activated by local injury [28].

BMAC with a Hyaluronic scaffold (Hyaff) has been used to treat chondral lesions of the knee with good clinical outcomes at long-term follow-up using [29–31]. This treatment was not only successful for single lesions but also in cases of multiple compartment injury, extensive lesions, or in older patients [30, 32, 33]. The composition of BMAC and what could be its mechanism of action has been a point of interest lately. Preliminary data seem to indicate there was no correlation between the clinical outcome and the number of Colony Forming Units (CFU; indirect estimation of the number of MSCs) found in bone marrow aspirate [29]. Interestingly, bone marrow aspirate harvested with Marrow Cellution system was shown to contain a relatively high CFU-fs/mL and CD34+/mL and therefore not requiring centrifugation. The level of CFU-fs/mL was significantly higher in comparison to BMAC in side-by-side evaluation from the same patient [34].

Although the use of BMAC shows good effects in cartilage repair surgery, its use as an injectable therapy is not so common. As a recent RCT has shown, there was no superiority of BMAC over PRP in knee OA therapy [35]. Though BMAC is one of the most appealing sources for cartilage defect repair, several aspects such as safety, amount of aspirate, and scaffold requirement require further investigation.

*Bio-Orthopedics: A New Approach to Osteoarthritis and Joint Disorders DOI: http://dx.doi.org/10.5772/intechopen.110845*

**Figure 2.** *Lipoaspirate transferred into ACP double syringe.*

#### **Figure 3.**

*Injection of the final product, 5 mL of leucocyte-poor platelet-rich plasma (LP-PRP) and 3 mL of SVF in a 10-mL syringe.*

#### **3. Applications**

#### **3.1 Ligament repair**

After a ligament is injured, a cascade of processes begins, it is characterized by cellular proliferation, migration, and collagen production. However, the process of platelet fibrin clot formation is significantly deficient in the cases of intra-articular injury [36]. Synovial fluid has been shown to reduce ACL fibroblast proliferation and migration, thus delaying tissue healing [37]. What is more, the presence of circulating plasmin in the joint space has been suggested to be the cause of suboptimal clot formation [38]. Without clot, the torn ligamentous fibers remain separated after injury and subsequent tissue repair is impaired.

PRP contains growth factors that enhance the processes related to cellular activity and their arrival at the area of the lesion. Platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) stimulate cell population's activation, migration, and proliferation. Fibroblast growth factor (FGF) is vital in collagen synthesis stimulation and fibroblast proliferation, both important elements in the tendon structure [39]. Due to these biological properties, PRP has been suggested to enhance the healing processes in ligaments.

For example, one study showed that the addition of PRP to the sutured ACL repairs did not improve AP knee laxity, maximum tensile load, or linear stiffness of the ACL after 14 weeks in vivo, compared to ACL repair without the addition of PRP [40]. However, when used with a collagen-PRP scaffold, the same author reported a significant improvement in load at yield, maximum load, and linear stiffness at four weeks. The PRP effect alone in ACL repair remains debatable, with significant graft maturation enhancement over time but no clear benefits on clinical outcomes [39].

Mixed results were also found with PRP administration in MCL lesions. LaPrade et al. described that one dose of either PPP or increasing twice the amount of PRP injected at the time of injury did not enhance ligament healing [41]. Furthermore, authors found that a four-time fold increase in the administered dose of PRP showed a significant adverse outcome on ligament strength six weeks after injury. In contrast, in a biomechanical analysis study by Yoshioka et al., there was a significant increase in the structural properties of MCLs in rabbits given leukocyte-poor PRP relative to controls [42].

Other cell therapies reported for ligament healing enhancement included dermal fibroblasts, which were shown to have promising properties in an in vivo models [43]. Adult MSCs represent a known cellular therapy used for tendon engineering; these cells' self-renewal capacity and multi-lineage differentiation potential have become common treatment. Bone marrow stromal cells embedded on polylactide or glycolide sutures have shown higher collagen production and DNA content than sutures seeded with anterior cruciate ligament fibroblasts and skin fibroblasts [41].

Combinations of cellular isolates and scaffolding have a promising role in treating tendon and ligament injury. In a prospective case series involving 50 patients (mean age 28.3 years), it was shown that ACL primary repair combined with bone marrow stimulation and BMAC-Hyaluronic acid bioabsorbable scaffold is an efficient method to restore knee stability and function in young athletes with acute partial ACL tears, after 10-year of follow-up [44, 45].

#### **3.2 Meniscal repair**

Even though techniques of repair have changed, many new instruments have been developed, still in the literature the failure rate of meniscus repair is reported to be around 20–25% [46]. Biological healing enhancement for the meniscal lesions treatment may be one of possible options to alter the outcomes [47].

Possible options for biological enhancement of menisci healing include rasping, needling, using a fibrin clot, platelet-rich plasma, bone marrow aspirate, and a scaffold with bone marrow addition [48].

In the 2019 ESSKA consensus on traumatic meniscus tears rasping, needling, and fibrin clot weak evidence was reported [48]. Although similar conclusion was reached in case of PRP use, a study that was not included in the consensus shown promising results. A prospective, randomized, double-blind, placebo-controlled trial with 37 patients with unstable complete vertical longitudinal tears, half treated with repair and placebo and half with repair and PRP. Same suturing all-inside technique was used. At 18-month follow-up the healing rate of tears was superior in the PRP-treated group (85% versus 47%) [49].

In another study on 550 patients treated for meniscal tears, authors found that the use of PRP resulted in improved survival of isolated meniscal repairs, but had no effect on survival of meniscal repairs with coexisting ACL reconstruction [50].

Bone marrow represents a great potential in healing enhancement. A prospective, randomized, double-blind, parallel-group, placebo-controlled study analyzing complete vertical meniscus tears in 40 patients randomized into two groups both using same suturing technique but one with addition of bone marrow venting procedure. Interestingly, the authors found a significant improvement of healing rate (rated by Second-Look Arthroscopy) in the bone marrow venting group compared to repair alone [51]. Another interesting study by Piontek et al. involved the use of meniscus suture with a collagen membrane wrapping together with bone marrow aspirate to treat combined and complex meniscal tears. They found a statistically significant improvement in subjective scores between the preoperative, 2-year follow-up, and 5-year follow-up [52, 53].

#### **3.3 Healing the cartilage**

Bone marrow stimulation techniques refer to methods using bleeding from subchondral marrow space and further formation of fibrin clot, which functions as scaffold for subchondral stem cell migration and consequent formation of fibrocartilage. In general, full-thickness cartilage lesion of a surface area < 1 cm2 is considered an indication for a bone marrow stimulation technique as an isolated procedure [54]. Although, one should be aware that these recommendations should be carefully considered for every patient individually.

Microfracture is most commonly known and used procedure due to availability, simplicity, and small cost [55–57]. The lesion should be prepared, loose cartilage should be removed, and borders made perpendicular to subchondral bone and then holes should be made with an arthroscopic awl. As the saline pressure is lowered, the release of fat droplets and the bleeding begins which will later form a clot on the defect [56, 58, 59]. Studies have shown that this fibrocartilage matrix consists mainly of type I collagen and other non-collagenous proteins, making this tissue more delicate and less elastic, that is why it is common for the initial satisfactory results to deteriorate over long term [60–62]. Better results may be expected in younger patients with smaller lesions. This technique should be used cautiously as it may damage the

subchondral bone and lead to the formation of microcysts, therefore, compromising the articular surface for future procedures [63].

Autologous Matrix-Induced Chondrogenesis (AMIC) is based on the same idea as microfracture but supported with a porcine collagen scaffold [64]. This technique is indicated for focal chondral or osteochondral defects, Outerbridge classification grade 3–4 with a defect size of 1.0–8.0 cm2, and patient age of 18 to 55 years old [3]. After the defect is treated with microfracture a scaffold is added to cover the lesion and to allow the ingrowth of mesenchymal stem cells (MSCs) from the subchondral bone. The main advantage of the AMIC procedure is no donor site morbidity and the possibility of arthroscopic approach. The procedure is also inexpensive compared to autologous chondrocyte implantation (ACI). Good clinical results of AMIC in midterm follow-ups have been described [65].

In acute lesions, use of autologous cartilage is an optimal alternative to repair a cartilage defect. It is described that covering an acute cartilage defect with minced fragments from a large piece of cartilage achieves good clinical results [66]. In this technique a large chondral fragment is minced into multiple small ones (<1 x 1 x 1 mm) with a scalpel. First, the cartilage lesion is debrided and drilled into the subchondral bone using a 1.4 mm K-wire. Then, minced cartilage fragments are placed into the defect and attached using fibrin glue. This concept is known since 1980s. The procedure using minced cartilage was modified and combined with various materials to become Cartilage Autograft Implantation System (CAIS) [67]. A cartilage paste (smaller cartilage size) was demonstrated to significantly increase extracellular matrix production [68]. Recently 10–23-year long-term results were reported in 74 patients cured with Articular Cartilage Paste Graft, the biopsies of the healed tissue revealed that 14 (48.3%) contained hyaline-like cartilage, 24 (82.8%) fibrocartilage with GAG, 10 (34.5%) fibrocartilage without GAG, and 3 (10.3%)fibrous tissue [69].

Osteochondral Autograft Transfer System (OATS) may be done in a single-stage procedure, arthroscopically or through arthrotomy. Cylindrical plugs are collected from donor sites from non-articulating regions. The plug consists of not only cartilage but also the subchondral bone, that is why it may recreate the osteochondral unit in cartilage lesions with damaged subchondral bone. This method is one of the few that has the advantage of restoring the hyaline cartilage. OATS is usually used for lesions smaller than 2 cm2 . In a 17-year prospective multicentric study performed in 383 found good to excellent results in 91% of femoral mosaicplasty, 86% of tibial, and 74% of patellofemoral mosaicplasty [70]. However, Wu et al. have shown that osteochondral plugs protruding 1 mm caused drastically increased contact pressures within the joint. Additionally, treatment using the OAT technique is restricted by the availability of autologous tissue, as donor site morbidity is a concern if multiple grafts are harvested.