**7. 3D spine modelling for epidural training**

In order to simulate the whole epidural procedure a realistic user interface must be provided together with the flexibility of 3D visualization and haptic interaction. The 3D models for the epidural simulator were generated with an object modelling software. Each vertebra is an individual wireframe model, constructed from 514 vertices. The vertices are positioned and then wrapped by a texture. Shadows and light sources are applied through OpenGL interfaces. The spine in the simulator contains 26 separate objects for the thoracic, cervical and lumbar spinal vertebrae, sacrum and coccyx. Layers of tissue, fat, muscle and

skin were appended as layers above the bones. The different parts of the model were exported into separate format files. The format is text based with each vertex on a separate line. A custom C++ OpenGL graphics application then parses the text file to re-create each vertex. The epidural Tuohy needle was created as separate 3D models allowing it to be moved around independently. This is important to allow the operator to place the needle anywhere along the spine for training purposes.

Biomedical Engineering in Epidural Anaesthesia Research 399

enable the palpation of spinal processes and identification of the midline. Finally, the sitting position combined with maximal lumbar flexion is also used, and having the patient bend forward is advantageous to the anaesthetists because it increases the space between the

Based on this, the patient could bend the spine to various positions, so the epidural simulator is required to use computer graphics models of the human spine which can bend, flex and twist. The model can realistically duplicate the shape of the spine during various sitting positions adopted by patients during surgery and epidural anaesthesia. The extent of bending and flexing is kept within the limits of human spine flexibility. Also the model vertebrate adapt in size to match weight and height of specific patient bodies based on parametric modelling [16]. Our spine model is flexible for epidural simulation which offers

The human spine consists of twenty six vertebrae. Each of the vertebrae connects with numerous ligaments. Internally, there is a protective space running through the centre of the spine, housing the spinal cord. The column of vertebrae also provides connection points with the ribs and back muscles. The twenty six vertebrae are segmented into five regions, each with varying characteristics. From cranial to caudal there are cervical vertebrae (C1- C7), thoracic vertebrae (T1 – T12), lumbar vertebrae (L1 – L5), sacrum and coccyx. The human spine is able to bend, flex and rotate in various directions. Lumbar flexion occurs when the patient bends forwards and lumbar extension occurs when bending backwards. The spine was modelled using 3D design software, formed from 26 individual vertebrae, shown in Figure 12. The 26 vertebrae were each loaded as 3D models into a custom made software graphics application. The software renders 3D objects using vertices with the OpenGL graphics library and its utility toolkit (GLUT). The colours of each region of

vertebrae, which increases the target space for the needle to pass through.

**Figure 11.** Four common patient positions used for epidural insertion

vertebrae bone, flesh and the spinal discs were set using materials.

**Figure 12.** The model spine consisting of 26 individually rendered 3D vertebrae

accurate models of spinal vertebrae.

The 3D objects can be viewed as stereograms (Figure 10) by displaying two images of the same object side by side with slight rotation around the Y axis [14]. The epidural simulator also supports this method of stereo in addition to page-flip stereo.

**Figure 10.** Stereogram view of the spine model with two perspectives and binocular parallax

Transparency is applied to skin, subcutaneous fat, supraspinous ligament, interspinous ligament and ligamentum flavum. This allows the user to see the position of the needle tip in the tissue layers. Transparency can be adjusted during the simulation by a control on the keyboard. Rotation is enabled allowing the camera angle to rotate around the scene. This is applied by OpenGL translation and rotation which gives an effect of camera movement whilst the other objects all remain stationary. During rotation, the tip of the needle remains at the central focus point of the screen. Zoom can be applied to move closer or further away from the site of insertion in the working epidural simulator. Pan can also be applied which is a translation of the camera which allows the user to view other areas or to move up and down the spine when selecting the insertion site.

Another issue equally important is the flexibility built into the spine model. There are four common patient positions adopted during the administration of spinal or epidural anaesthesia [15]. Lateral decubitus (Figure 11) involves lying down sideways on the patients left or right, usually the right side is used for caesarean patients, because it is the opposite side from which the patient will lie on during surgery in the left lateral tilt position, which helps to increase the spread of anaesthetic. When the patient lies in lateral positions their back should be close and parallel to the edge of the bed, with their spine in a straight line. However, a variation to this position, maximal lumbar flexion in the lateral decubitus position can be used. The sitting position is preferred and often required in obese patients to enable the palpation of spinal processes and identification of the midline. Finally, the sitting position combined with maximal lumbar flexion is also used, and having the patient bend forward is advantageous to the anaesthetists because it increases the space between the vertebrae, which increases the target space for the needle to pass through.

**Figure 11.** Four common patient positions used for epidural insertion

398 Practical Applications in Biomedical Engineering

anywhere along the spine for training purposes.

down the spine when selecting the insertion site.

also supports this method of stereo in addition to page-flip stereo.

skin were appended as layers above the bones. The different parts of the model were exported into separate format files. The format is text based with each vertex on a separate line. A custom C++ OpenGL graphics application then parses the text file to re-create each vertex. The epidural Tuohy needle was created as separate 3D models allowing it to be moved around independently. This is important to allow the operator to place the needle

The 3D objects can be viewed as stereograms (Figure 10) by displaying two images of the same object side by side with slight rotation around the Y axis [14]. The epidural simulator

**Figure 10.** Stereogram view of the spine model with two perspectives and binocular parallax

Transparency is applied to skin, subcutaneous fat, supraspinous ligament, interspinous ligament and ligamentum flavum. This allows the user to see the position of the needle tip in the tissue layers. Transparency can be adjusted during the simulation by a control on the keyboard. Rotation is enabled allowing the camera angle to rotate around the scene. This is applied by OpenGL translation and rotation which gives an effect of camera movement whilst the other objects all remain stationary. During rotation, the tip of the needle remains at the central focus point of the screen. Zoom can be applied to move closer or further away from the site of insertion in the working epidural simulator. Pan can also be applied which is a translation of the camera which allows the user to view other areas or to move up and

Another issue equally important is the flexibility built into the spine model. There are four common patient positions adopted during the administration of spinal or epidural anaesthesia [15]. Lateral decubitus (Figure 11) involves lying down sideways on the patients left or right, usually the right side is used for caesarean patients, because it is the opposite side from which the patient will lie on during surgery in the left lateral tilt position, which helps to increase the spread of anaesthetic. When the patient lies in lateral positions their back should be close and parallel to the edge of the bed, with their spine in a straight line. However, a variation to this position, maximal lumbar flexion in the lateral decubitus position can be used. The sitting position is preferred and often required in obese patients to Based on this, the patient could bend the spine to various positions, so the epidural simulator is required to use computer graphics models of the human spine which can bend, flex and twist. The model can realistically duplicate the shape of the spine during various sitting positions adopted by patients during surgery and epidural anaesthesia. The extent of bending and flexing is kept within the limits of human spine flexibility. Also the model vertebrate adapt in size to match weight and height of specific patient bodies based on parametric modelling [16]. Our spine model is flexible for epidural simulation which offers accurate models of spinal vertebrae.

The human spine consists of twenty six vertebrae. Each of the vertebrae connects with numerous ligaments. Internally, there is a protective space running through the centre of the spine, housing the spinal cord. The column of vertebrae also provides connection points with the ribs and back muscles. The twenty six vertebrae are segmented into five regions, each with varying characteristics. From cranial to caudal there are cervical vertebrae (C1- C7), thoracic vertebrae (T1 – T12), lumbar vertebrae (L1 – L5), sacrum and coccyx. The human spine is able to bend, flex and rotate in various directions. Lumbar flexion occurs when the patient bends forwards and lumbar extension occurs when bending backwards. The spine was modelled using 3D design software, formed from 26 individual vertebrae, shown in Figure 12. The 26 vertebrae were each loaded as 3D models into a custom made software graphics application. The software renders 3D objects using vertices with the OpenGL graphics library and its utility toolkit (GLUT). The colours of each region of vertebrae bone, flesh and the spinal discs were set using materials.

**Figure 12.** The model spine consisting of 26 individually rendered 3D vertebrae

Initially the vertebrae are positioned in the standing position and are then adjusted by mathematical equations to match the current patient position. The curvature of the spine for four common patient positions was calculated using the equations. The shape of the spine was based on the four common patient positions used for epidural insertion. Our model's prediction for the spine shape for each of the positions is shown in Figure 13 [14].

Biomedical Engineering in Epidural Anaesthesia Research 401

As LF thickness increases, fibrosis increases and elastic tissue decreases. The dorsal side of LF contains more fibrous tissue and less elastic tissue than the dural and middle sides, as indicated by a fibrosis Score of 1.58, 1.63, and 2.63 for dural, middle, and dorsal sides respectively [18]. The loss of elastic fibres caused by increased thickness is more pronounced along the dorsal side. A single patient has several ligamentum flava, one at each spinal level between the lamina and their thicknesses vary according to the spinal level. A study of 77 patients measured LF at spinal level L2/3, L3/4, L4/5, and L5/S1, the mean LF thickness is 2.41, 3.25, 4.08, and 2.68 mm [18]. It was shown that the thickest part of ligamentum flavum is consistently at L4/5, which is the level that endures the greatest mechanical stress. LF is crescent shaped in cross section on the horizontal plane with the thickest part in the middle. It wraps around the circular epidural space and dura. It connects to lamina above and below. The elastic fibres are yellow in colour, hence 'flava' being Latin for yellow. Each flava

Object modelling software was used to create a model of the vertebrae. At the location of L2/L3 a ligamentum flavum was modelled with the thickness 2.41mm which was internally

The interior structure of the ligamentum flavum has been modelled by numerous bundles of fibres extending vertically and parallel to one another, as do the elastic and fibrous tissues in-vivo. By creating this heterogeneous model of the internal structure of ligamentum flavum, the model will describe more accurately how the material responds to a needle being inserted through it. Similar models may be created for interspinous ligament and supraspinous ligament which are also both heterogeneous in nature, consisting of over three

We have applied stereoscopic 3D computer graphics for visualization of epidural insertions.  The 3D graphics are built from several vertex models of the anatomical structures as described in section 7. The stereo

is a separate ligament which is clearly seen from the side of the lamina.

**Figure 14.** The modelled ligamentum flavum between L2/L3 vertebrae.

types of elastic fibres that can used to provide realistic haptic feedback.

**9. 3D visualisation of epidural procedure**

comprised of bundles of fibres (Figure 14).

**Figure 13.** The spine model with flexion for four common patient positions

The ability to flex and rotate the spine has provided the opportunity to simulate epidural insertions on patients in various positions. This is important because the feeling of insertion is different for each patient position. This novel aspect has not been attempted in epidural simulation before and will increase versatility of the simulation.


**2. Dermoscopy**

Dermoscopy of the nail unit is a noninvasive method that can help identify highrisk features.

Dermoscopy is useful to distinguish blood; subungual hemorrhage has a distinctive pattern of globules with distal streaks, a filamentous end, and red to brown or deep purple color. It is important to consider a bleeding tumor and rule out that possibility [5].

#### **Figure 3.**

*Dermoscopy of SUM in situ. Irregular multiple heterogenous brown bands with blurred edges and microhutchinson's sign.*

Subungual melanoma should be suspected and ruled out in heterogeneous longitudinal brown or black melanonychias, when bands are irregular in color, thickness, and spacing. SUM can also present as a diffuse dark background with barely visible lines (**Figure 3**). When a brown coloration in the background is overlaid by regular, parallel, and pigmented lines, the most probable diagnosis is a nevus.

Edge blurring is another sign associated with SUM. Hutchinson's sign is considered an indicator of SUM; however, it can also be found in benign nevi. Atypical Hutchinson's sign in SUM is asymmetric and polychromatic, and the pigment is distributed in a disorderly fashion. Micro-Hutchinson's sign is periungual pigmentation invisible to the naked eye and only observed with dermoscopy; it has only been described in SUM. Triangular shape of the longitudinal band (wider proximally than distally) indicates rapid growth [5, 6].

A grayish longitudinal background either alone or overlaid by thin homogenous gray lines is suggestive of melanocytic hyperplasia as in lentigo or lentiginoses (Laugier-Hunziker syndrome, Leopard syndrome, Peutz-Jeghers-Touraine disease), in drug-induced, ethnic, and traumatic nail pigmentation.

Amelanotic SUM is a very difficult diagnosis; in this rare case, the nail plate is often partially destroyed by a bleeding, erythematous vegetating tumor. Dermoscopy can show areas of remanant pigmentation and vascular disorder: irregular vessels and milky-red areas [5].


**1. Introduction**

For over a century, plant tissue culture technology has been an important tool in crop improvement and development: producing disease-free plant material [1], obtaining transgenic plants [2,3], breaking dormancy, and micropropagating elite plants with highly desirable chemotype [4], thereby leading to more uniform plant production [5,6]. This is the technology for conserving in vitro germplasm of elite [7,8], rare, and endangered plant species [9–11], implementing breeding programs for innumerous crops as well as encapsulated seeds [12], and studying plant biosynthesis through cell and root cultures [12,13], the interaction between endophytes and the hostplant [14,15].

High-demand plants face great challenges: Depletion of species diversity due to overharvesting and environmental pollution affecting natural populations are strong factors that support the argument for cultivating rare and elite high-yielding medicinal plants. In addition, the cultivation of medicinal plants is the most effective way of addressing the gap between supply and demand. Breeding studies are necessary both to develop pharmaceutical plants as crops and to scale up their production [16]. Still, few success stories about breeding medicinal plants such as *Artemisia annua* L. exist. Because micropropagation is the tool of producing clones—especially with high-yielding chemotypes—for industrial purposes, it solves this target-breeding problem. Moreover, as the *Echinacea* study [17] showed, micropropagation's demonstrated ability for mass selection suggests that together with bioassays it could form part of an overall strategy to screen elite phenotype lines.

**Citation:** Moraes, R.M.; Cerdeira, A.L.; Lourenço, M.V. Using Micropropagation to Develop Medicinal Plants into Crops. *Molecules* **2021**, *26*, 1752. https:// doi.org/10.3390/molecules26061752

Academic Editors: Muhammad Ilias and Dhammika Nanayakkara

Received: 24 February 2021 Accepted: 19 March 2021 Published: 21 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Micropropagation is an in vitro technology of rapidly multiplying elite plants using modern plant tissue culture methods. It is well-known for its applications in the agro, horticultural and forestry industries, this review focuses on a less-commonly known area which is on medicinal plants and the need to develop them as medicinal crops. Li et al. [18] defined pharmaceutical crops in three distinct categories: 1) crops for the production of small therapeutic molecules (STMs), 2) standardized therapeutic extracts (STEs), and 3) large therapeutic molecules (LTMs). In addition, this review also examines micropropagation of functional food plants to ensure their development as crop.

### **2. Pharmaceutical Crops for Production of Small Therapeutic Molecules (STM)**

Drug discovery programs and the formation of knowledge of different pharmacological classes of pharmaceuticals owe much to traditional medicine in countries such as China and India [19,20]. Some natural compounds are extracted or used as templates for synthesis or as a precursor for the semi-synthesis (e.g., paclitaxel, artemisinin, podophyllotoxin, cannabinoids, galantamine, vinca alkaloids, atropine, ephedrine, digoxin, morphine, quinine, reserpine, tubocurarine etc.). Many of these compounds provide therapeutic relief for several major illnesses including cancer, Alzheimer, malaria, high blood pressure, fever, and anxiety. As researchers confirm the medicinal utility of these natural resources, they suffer depletions with the increased demand.

According to McChesney et al. [20], pharmaceutical natural substances require considerations beyond supply and demand: the establishment of successful production systems must be sustainable, environmentally safe, reliable, and affordable. Thus, the development of medicinal crops is a key factor to obtaining a commercially viable source of medicinal biomass for the pharmaceutical industry. In fact, non-stable supply sources could lead to bottlenecks that limit potentially beneficial products. For example, researchers pointed to insufficiency in the biomass supply of anti-cancer pharmaceutical ingredients such as podophyllotoxin and paclitaxel, as the major limiting factor at phase III clinical trials, which led to overharvesting of the natural resources of *Podophyllum emodi* Wall ex Royle in India [21] and *Taxus baccata* L. in Europe [22].

Given the shortage of biomass supply limiting clinical phase III trials of paclitaxel and podophyllotoxin, several laboratories engaged in different research approaches that included bioprospecting studies searching for alternate sources with high yields of the active compounds [23–25]. Clippings of cultivated *Taxus* sp. became the reliable source for production of paclitaxel [18], and Sisti et al. [26] reported methods of semi-synthesis using abundant intermediates for production of paclitaxel. Majada et al. [27] reported a procedure to obtain high-yielding *T. baccata* plantlets by screening micropropagated juvenile seedlings that accumulate 10-deacetyl baccatin III. The selected genotypes of *T. baccata* grow faster and contain high taxene content.

For its part, podophyllotoxin is the starting compound for semisynthesis of etoposide and teniposide, two potent DNA topoisomerase cancer drugs utilized in the treatment of small lung and testicular cancers, lymphomas/leukemias and the water-soluble etoposide phosphate, also known as etopophos (Figure 1). To assure podophyllotoxin supply, a buffer extraction procedure using leaf biomass of mayapple plants provides a sustainable alternative source [28]. Later, we published a survey and a database of high-yielding podophyllotoxin colonies [29,30] and an in vitro propagation protocol of Podophyllum peltatum L. to rapidly produce podophyllotoxin-rich plantlets [5].

**Figure 1.** Structures of (−)-podohyllotoxin present in *Podophyllum* sp and its commercial chemotherapeutic derivatives.

*Artemisia annua* L. is the source of artemisinin, an endoperoxide sesquiterpene lactone that is very difficult to synthesize, precursor of a common anti-malarial drug (Artemether). Artemisinin production comes from cultivated plants selected for their high artemisinin content [18]. Selection of genotypes with high artemisinin concentration in wild populations resulted in lines containing up to 1.4 percent artemisinin (on dry leaves basis). The leading commercial source, 'Artemis,' exhibited extensive variation of metabolic and agronomic traits; artemisinin content on a μg/mg dry basis for individual plants ranged 22-fold, plant fresh weight varied 28-fold, and leaf area ranged 9-fold [31].

While Ferreira and Janick [32] found that the in vitro production of artemisinin will never be commercially feasible, Wetzetein et al. [33] suggested that cultivation of micropropagated high-yielding artemisinin plants with levels above 2% and improved agronomic traits (high leaf area and shoot biomass production) may reach productivity of 70 kg/ha artemisinin using a crop density of 1 plant m<sup>−</sup>2. We include in Table 1 examples of pharmaceutical plant species classified as small therapeutic molecules STM's (18) and their micropropagation protocols to produce elite clones for higher yields. Taxol® is another success story. According to McChesney et al. [20], the path from the discovery to a pharmaceutical drug requires a viable production system (cultivation, harvest, extraction, purification and isolation) where every step of a natural product must be systematically evaluated. Micropropagation of the superior source (chemotype or variety of the species) may help

to produce biomass with a high and consistent concentration of the natural product or a precursor of the natural product that can be converted economically by semi-synthesis to the final bulk active product.

**Table 1.** Commercial sources of pharmaceuticals often used in therapies of several illnesses that are micropropagated [17].

#### **3. Phytomedicine Crops for the Production of Standardized Therapeutic Natural Products**

Herein, we describe the category of phytomedicinal crops similar to what Li et al. [18] reports regarding pharmaceutical crops for production of standardized therapeutic extracts (STEs). Additionally, we relate examples wherein micropropagation proves useful as a method for ensuring the stability of biomass supply of phytomedicines by allowing breeders to select phytomedicinal crops with an eye towards maintaining genetic consistency and the sustainability of wild plant population.

Also known as botanical drugs, herbal remedies, and herbal medicines, phytomedicines are classified in the United States as dietary supplements according to the specific claim as described in the Dietary Supplement Health and Education Act (DSHEA) of 1994 [18]. In Europe, the phytomedicines are standardized and certified medicinal products and in Asia they have a status of traditional medicine. Phytomedicinal crops relate to the cultivation of medicinal species by which a mixture of multiple active compounds commercialized as standardized products. Usually, phytomedicines are evaluated for quality as the means to ensure safety, as complex mixtures of secondary compounds, to maintain consistency is fundamental to their efficacy. Thus, authenticity and uniformity and well-defined cultivation practices and postharvest processes are essential to certify safety and efficacy. Govidaraghavan and Sucher [44] reinforce that herbal productions must follow good agricultural and collection practice (GACP), good plant authentication and identification practice (GPAIP), good manufacturing practice (GMP) before and during the manufacturing process, guided by analytical tools, and micropropagation is an important tool in ensuring uniformity and consistency in open pollinated crops.

As of today, the majority of phytomedicines are still harvested from the wild, which causes habitat destruction, genetic diversity loss, as well as ingredient mislabeling, variability and contamination. In Brazil, products are sourced from the wild, as well as from cultivation in agroforest or in small gardens. They are chosen without proper guide from health-care professionals because medical schools do not include in their curriculum the disciplines of phytomedicines or phytotherapy. In 2016 the Brazilian Health Regulatory Agency, ANVISA, officially recognized twenty-eight medicinal plant species as herbal drugs and published their monographs [45] in the first edition (Memento). The monographs are a complete therapeutic guide of phytomedicinals reviewed and accepted by ANVISA as therapies used in SUS, the public health system of Brazil. The majority of the phytomedicinals included in this first edition, was introduced to Brazil by immigrants and later became part of traditional use especially by the rural communities.

The increased consumption of phytomedicine offers an opportunity to develop medicinal plant production systems as crop. Conventional plant breeding may improve agronomic traits in association with molecular markers aiding crop development. The greatest obstacles for such a program remain predicting which extracts remain active, specifically resembling all the medicinal properties described in the ones harvested in the wild [46]. In this context, micropropagation may produce clones that could be screened using bioassays to assure bioactivity. Moraes et al. [17] used tissue culture techniques to produce *Echinacea* sp. clones and later screened those using human monocytes assays to identify high and low activity. The immune response between the two selected clones after field cultivation due to bacterial endophytes was the same [47]. The selection procedure using in vitro propagation techniques, genetic markers, and bioassay work are approaches for selection of elite germplasm [17].

Micropropagation allows one to mass generate plants with genetically identical chemotype for cultivation purposes. Reinhard [48] suggested that different chemotypes in Cat's Claw (*Uncaria tometosa* (Willd. ex Schults) DC) might have different healing properties: tetracyclic oxindole alkaloid acting on the central nervous system, and the pentacyclic oxindole alkaloid affecting the immune system. The immunological effect of both alkaloid mixtures is antagonistic and therefore may be unsuitable for therapy. For Reinhard [48], the production of safe and efficacious Cat's Claw phytomedicinal requires chemical identification prior to harvesting and perhaps even before the cultivation.

Micropropagation also allows one to select plants based on the chemical profile in order to standardize a particular chemotype. Morais et al. [49] reported that the chemical composition of *Lippia sidoides* Cham. (syn. *Lippia origanoides)* varied according to cultivation sites. Thymol is the major component of essential oil extracted from crops grown in northeast Brazil [50–52], whereas carvacrol is the major component present in *L. sidoides* harvested from Lavras, Minas Gerais [53] and 1.8-cineole, isoborneol, and bornyl acetate in São Gonçalo do Abaeté, Minas Gerais, Brazil. Standardized essential oil of *L. sidoides* is recommended for topical applications on skin, mucous membranes, mouth, throat and vaginal washings as antiseptic [45]. According to Santos et al. [53], genotypes regulate chemical polymorphism thymol and carvacrol. Phenotypical variation is likely to influence biological properties and the type of industrial application. Planting thymol or carvacrol clones ensured a high-quality biomass for safe and efficacious products [54].

Finally, micropropagation proves useful to reduce consumption pressure on potentially threatened wild populations [55]. For example, bark extraction of barbatimão to produce phytomedicine has depleted genetic diversity of *Stryphnodendron polyphythum* Mart. natural resources. The bark of this Brazilian tree is widely utilized as a woundhealing phytomedicine with anti-inflammatory, antioxidant and antimicrobial activities. Souza-Moreira et al. [55] showed that proanthocyanidins present in the bark are responsible for its healing properties. França et al. [10] published an efficient micropropagation protocol to produce barbatimão plantlets, while Correa et al. [56] defined the conditions for in vitro germplasm conservation to reduce pressure on its threatened status. Table 2 includes in vitro propagation protocols to produce healthy plantlets for cultivation purposes, thus aiding the development of phytomedicinal crops.

As the above paragraphs state, micropropagation can provide an effective technique to those seeking to mold a supply chain of a product, in order to ensure the genetic homogeneity of plant clones, chemical profile, and finally sustainability of those plants harvested in the wild.

**Table 2.** Micropropagation protocols of medicinal plants considered phytomedicine by the Brazilian Regulatory Agency (ANVISA).


*3.10. Statistical Analysis*

All the experiments were conducted three times and the data were presented as mean with standard error based on descriptive statistics. One way ANOVA (*p* < 0.05) and post hoc test were adapted using the statistical software SPSS version 16.0 (SPSS, Chicago, IL, USA).

### **4. Conclusions**

In this study, two types of collagens, PSC and ASC were isolated and purified from blue shark skin, which contained three distinct chains, 2 alpha, and beta chains. The amino acid composition and the FTIR secondary structure of PSC varied with ASC due to pepsin hydrolysis. Among two collagens, PSC had high thermal stability, which might be due to high imino acid content. Most importantly, the obtained collagens promoted osteoblast cell growth and upregulated collagen synthesis in bone cells, which is a most desirable property of biomaterials for the treatment of a bone disorder. Proliferation of differentiated osteogenic cells by blue shark skin collagen may be achieved through activation of Runx2 dependent FAK/JNK signaling pathway. Overall, the blue shark skin collagens with good biochemical and osteogenic properties may be considered as a potential drug in biomedical application. Conversely, the in vivo evaluation of blue shark skin collagen and its molecular interaction with bone cells needs to be addressed by further research in order to understand the actual mechanism of the action.

**Author Contributions:** J.E. and J.W.L. carried out the laboratory works, compiled data, and wrote the manuscript. Y.H., S.W., J.E.M.S.d.V., S.Y.L. and J.M.R. revised the manuscript and provided valuable advice on interpretation of data. W.W. and B.B. designed and directed the study, inferred the data, and revised the manuscript.

**Funding:** This research was funded by National Natural Science Foundation of China, project grant number<PHONE_NUMBER>8, the National High Technology Research and Development Program of China, project grant number 2011AA09070109), and the Plan of Innovation Action in Shanghai project grant number<PHONE_NUMBER>0 and<PHONE_NUMBER>0.

**Acknowledgments:** The authors gratefully acknowledge the funding support received from the National Natural Science Foundation of China (Project Grant No.<PHONE_NUMBER>8), an International Young Scientist Research Fellowship, the National High Technology Research and Development Program of China (Project Grant No. 2011AA09070109), and the Plan of Innovation Action in Shanghai (Project Grant No.<PHONE_NUMBER>0 and<PHONE_NUMBER>0). The authors also thank Shanghai Ocean University, Shanghai, China for providing the necessary facilities to undertake this work.

**Conflicts of Interest:** The authors declare no conflict of interest.