The use of this procedure results in an inconclusive recommendation strength [10].

#### **4. Hyaluronic acid injections**

HA plays a fundamental role in maintaining elasticity and viscosity of the synovial fluid and integrity of the connective tissues such as joints [40, 41]. Several studies have shown that HA is a chondroprotector: it synthesizes proteoglycan and glycosaminoglycan, and it has anti-inflammatory, mechanical, subchondral, and analgesic actions [40].

#### **5. Platelet-rich plasma**

Platelet-rich plasma (PRP) is the most investigated biological treatments [42–44] due to the capacity to reduce inflammation and consequently a reduction of pain [45–47]. PRP exerts its biological effect with neoangiogenesis and migration of macrophages and mesenchymal cells and regulates cell differentiation and the activity of different cell lines, promoting tissue regeneration. PRP is controversial for the treatment of OA, because there is insufficient evidence to recommend the use of it [48–50].

Despite the growing interest in this biological approach for cartilage regeneration, the knowledge on this topic is still preliminary [51].

**43**

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results*

The promise of mesenchymal stem cells (MSCs) to give birth to a new era of medicine was strong, thanks to the ability of these cells to do self-renewal and multipotent *in vitro* differentiation into mesodermal cell subtypes. To be honest, recent studies have demonstrated that a good portion of the thrilling *in vivo* clinical results is due to the trophic, paracrine, and immunomodulatory activities of MSCs instead of their differentiation ability [52]. Differently from drug concept where the effect is dependent from concentration, MSCs are self- and site-regulated and they release a multitude of bioactive factors in variable concentration in response to the local messages of the microenviroment. The main trophic activity exerted by MSCs is the release of growth factors and other chemokines to induce the homing and proliferation of cellular progenitors and to promote angiogenesis. These factors are transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), endothelial growth factor (EGF), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor 1 (IGF-1)—all of these are proteins able to accelerate cellular growth and division of progenitors [53]. Moreover, IGF-1, EGF, and the vascular endothelial growth factor (VEGF) are able to recruit endothelial cells and promote new vascularization [54]. MSCs can derive from many tissue sources, and among these, the more manageable for clinical practice is bone marrow and adipose tissue. Even if the MSCs from bone marrow (BM-MSCs) were discovered first and have more clinical experience, there is higher interest on adipose tissue—not only for the ease and low morbidity of harvest but also because it has a higher MSCs frequency. In a bone marrow aspirate,

/ml nucleated cells and, among these, only 0.001–0.01% are MSCs;

/g of MSCs, it means that there is a 500-fold higher

MSCs frequency ranges from 1 to 10% based on the donor site. Since the adipose tis-

concentration of MSCs in comparison with bone marrow [55]. Moreover, it has been demonstrated that the proliferation and differentiation properties of stem cells from adipose tissue (ADSCs) are less impaired by age in comparison with BM-MSCs [56]. ADSCs can be exploited with three different methods. The first, the only option where we can properly name them as true ADSCs, is the cell culture and optional cell expansion *in vitro*. By selecting the cells that are able to stick to the plastic, it is possible to obtain cell that expresses mesenchymal markers (CD105+, CD73+, CD90+, CD45−, CD34−, CD11b−, CD14−, CD79a−, and HLA-DR−) able to differentiate into the three mesodermal lineages (bone, cartilage, and fat). This option has serious limitation in the clinical practice due to regulatory issues because it can only be performed into good manufacturing practice facilities that manipulate these cells like an experimental drug (it is named advanced cellular therapy). Notably, concentrated BM-MSCs outperform cultured cells since they are more practical and efficient and less harmful and expensive [52]. The second method is enzymatic digestion of adipose tissue that gives in the hands of operators a heterogeneous cell population that contains, beside MSCs, endothelial cells, leucocytes, and preadipocytes. This final product of enzymatic digestion is named stromal vascular-fraction (SVF). Finally, autologous ADSCs can be exploited through the processing and fragmentation of adipose tissue (FAT, *fragmented adipose tissue*). While the SVF is a heterogeneous cell population where each cell is separated from the others and the efficacy is dose-dependent, the FAT is a proper minimally manipulated tissue that entrust more on cell quality instead of cell quantity. The FAT is composed of tissue cluster of variable diameter where the SVF cells are embedded and attached to an undisrupted tissue architecture made of vasculature and extracellular matrix sustained by a scaffold made of adipocytes. This natural scaffold protects cells from anoikis (death cause by the lack of cellular adherence) and other harmful stress.

/g nucleated cells where the

*DOI: http://dx.doi.org/10.5772/intechopen.82566*

**6.  Biological use of fat tissue**

*Osteoarthritis Biomarkers and Treatments*

the clinical condition of the patient.

activate a regenerative process into the joint.

mended [10, 34, 35].

after injection.

hyperglycemia [39].

analgesic actions [40].

use of it [48–50].

**5. Platelet-rich plasma**

**4. Hyaluronic acid injections**

knowledge of the disease and the consequent implementation of some measures such as weight loss and gymnastics or the use of unloading orthoses is recom-

For conservative treatment, today we have several strategies available that, as mentioned, where possible, must be evaluated and taken into account in relation to

An increasingly important role in the conservative treatment of OA is represented by the infiltrative therapy that in recent years has proposed a wider range of solutions: from intra-articular anti-inflammatory therapy as a palliative treatment to an infiltrative solution that can restore joint homeostasis or that can possibly

Intra-articular corticosteroid injections may be indicated after failing NSAIDs and acetaminophen, but some researchers suggest only using them once every 3 months for a maximum of 2 years due to negative potential side effects [36].

The mechanism underlying the anti-inflammatory efficacy of corticosteroid is multifactorial, but generally involves blocking antigen opsonization, leukocytic cell adhesion, and cytokine diapedesis within the capillary endothelium. Corticosteroids also attenuate the effects of IL-1, decrease leukotriene and prostaglandin release, and inhibit metalloproteases and immunoglobulin synthesis [37]. The duration of action of intra-articular corticosteroid injections remains controversial, with various studies quoting anywhere between 1 and 24 weeks. There is consensus that steroids provide relief to patients for approximately 1 week

Adverse effects of corticosteroid injections do exist; however, Handler and Wright first described radiographic evidence of destruction of the knee joint and cartilage after several corticosteroid injections [38]. The incidence of joint infection following corticosteroid administration is rare, but may be as high as one in 3000 patients, with an associated mortality rate of approximately 11%. Additional known complications include pain, skin atrophy, tendinopathy, and systemic

The use of this procedure results in an inconclusive recommendation strength [10].

HA plays a fundamental role in maintaining elasticity and viscosity of the synovial fluid and integrity of the connective tissues such as joints [40, 41]. Several studies have shown that HA is a chondroprotector: it synthesizes proteoglycan and glycosaminoglycan, and it has anti-inflammatory, mechanical, subchondral, and

Platelet-rich plasma (PRP) is the most investigated biological treatments [42–44] due to the capacity to reduce inflammation and consequently a reduction of pain [45–47]. PRP exerts its biological effect with neoangiogenesis and migration of macrophages and mesenchymal cells and regulates cell differentiation and the activity of different cell lines, promoting tissue regeneration. PRP is controversial for the treatment of OA, because there is insufficient evidence to recommend the

Despite the growing interest in this biological approach for cartilage regenera-

tion, the knowledge on this topic is still preliminary [51].

**42**

The promise of mesenchymal stem cells (MSCs) to give birth to a new era of medicine was strong, thanks to the ability of these cells to do self-renewal and multipotent *in vitro* differentiation into mesodermal cell subtypes. To be honest, recent studies have demonstrated that a good portion of the thrilling *in vivo* clinical results is due to the trophic, paracrine, and immunomodulatory activities of MSCs instead of their differentiation ability [52]. Differently from drug concept where the effect is dependent from concentration, MSCs are self- and site-regulated and they release a multitude of bioactive factors in variable concentration in response to the local messages of the microenviroment. The main trophic activity exerted by MSCs is the release of growth factors and other chemokines to induce the homing and proliferation of cellular progenitors and to promote angiogenesis. These factors are transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), endothelial growth factor (EGF), fibroblast growth factor 2 (FGF-2), and insulin-like growth factor 1 (IGF-1)—all of these are proteins able to accelerate cellular growth and division of progenitors [53]. Moreover, IGF-1, EGF, and the vascular endothelial growth factor (VEGF) are able to recruit endothelial cells and promote new vascularization [54].

MSCs can derive from many tissue sources, and among these, the more manageable for clinical practice is bone marrow and adipose tissue. Even if the MSCs from bone marrow (BM-MSCs) were discovered first and have more clinical experience, there is higher interest on adipose tissue—not only for the ease and low morbidity of harvest but also because it has a higher MSCs frequency. In a bone marrow aspirate, there are 6 × 106 /ml nucleated cells and, among these, only 0.001–0.01% are MSCs; on the other hand, a lipoaspirate contains 0.5–2.0 × 106 /g nucleated cells where the MSCs frequency ranges from 1 to 10% based on the donor site. Since the adipose tissue has a total of 0.5 × 104 –2 × 105 /g of MSCs, it means that there is a 500-fold higher concentration of MSCs in comparison with bone marrow [55]. Moreover, it has been demonstrated that the proliferation and differentiation properties of stem cells from adipose tissue (ADSCs) are less impaired by age in comparison with BM-MSCs [56].

ADSCs can be exploited with three different methods. The first, the only option where we can properly name them as true ADSCs, is the cell culture and optional cell expansion *in vitro*. By selecting the cells that are able to stick to the plastic, it is possible to obtain cell that expresses mesenchymal markers (CD105+, CD73+, CD90+, CD45−, CD34−, CD11b−, CD14−, CD79a−, and HLA-DR−) able to differentiate into the three mesodermal lineages (bone, cartilage, and fat). This option has serious limitation in the clinical practice due to regulatory issues because it can only be performed into good manufacturing practice facilities that manipulate these cells like an experimental drug (it is named advanced cellular therapy). Notably, concentrated BM-MSCs outperform cultured cells since they are more practical and efficient and less harmful and expensive [52]. The second method is enzymatic digestion of adipose tissue that gives in the hands of operators a heterogeneous cell population that contains, beside MSCs, endothelial cells, leucocytes, and preadipocytes. This final product of enzymatic digestion is named stromal vascular-fraction (SVF). Finally, autologous ADSCs can be exploited through the processing and fragmentation of adipose tissue (FAT, *fragmented adipose tissue*). While the SVF is a heterogeneous cell population where each cell is separated from the others and the efficacy is dose-dependent, the FAT is a proper minimally manipulated tissue that entrust more on cell quality instead of cell quantity. The FAT is composed of tissue cluster of variable diameter where the SVF cells are embedded and attached to an undisrupted tissue architecture made of vasculature and extracellular matrix sustained by a scaffold made of adipocytes. This natural scaffold protects cells from anoikis (death cause by the lack of cellular adherence) and other harmful stress.

The MSCs embedded into these clusters have high vitality and can still differentiate into the three mesodermal lineages as a proof of their multipotency. To prove that quality is superior to quantity, it has been demonstrated in an animal model of critical limb ischemia that an injection of 500 μl of FAT (which contain 2 × 104 ADSC) restores limb perfusion better than a single injection of 1 × 106 isolated ADSC [57].

During the First World War, the fat was used to heal soldier wounds and its application was very heterogeneous until 1990 where Sidney Coleman defined precise guidelines. The evolution of surgical techniques associated the volumizing effect of lipofilling to the regenerative properties of natural adipose tissue, favoring the processing, the reduction, and the purification of the tissue to raise the survival of grafts and magnify the regenerative potential of MSCs. Also reducing the volume of grafts and the diameter of injection needles had helped the hosting tissue to decrease stress and trauma, thus provoking less inflammation and ameliorating again graft survival [58]. Adipose tissue is now used with lipofilling techniques in hard-toheal wounds and in all cases characterized by mortified tissue (burn, compression, and radiation) linking the regenerative potential also to an esthetic effect. Wound healing professionals use lipofilling to treat ulcers resistant to classical therapies and advanced dressing and also for the treatment of critical limb ischemia and diabetic patients [59]. In orthopedics, intra-articular lipofilling has become a fashionable and innovative strategy to fight osteoarthritis, thanks to the ability of this tissue product to reequilibrate the articular homeostasis and to reduce the inflammation of the synovial membrane [60]. The integrity of the extracellular matrix can also exploit the shock absorber function of adipose tissue reducing the stress between cartilage surfaces. The paracrine effect of MSCs can also promote cartilage repair when mechanical conditions of the articulation are stable. The anti-adhesive properties of adipose tissue were exploited in the surgery of tendons and nerves to limit the formation of fibrotic and scarring tissue, thus also limiting the relapse incidence. Finally, adipose tissue was also studied in pain management for the intradiscal infiltration for the treatment of low back pain associated to black disc.

#### **7. Lipofilling**

The technique of liposculture is originally created for esthetic purposes. Autologous fat transfer has recently become an increasingly popular surgical procedure: harvesting, refinement/processing, and transfer of subcutaneous tissue to provide relatively pure and intact parcels of fat are paramount for successful lipofilling. Usually, we use local anesthesia with sedation or epidural anesthesia. Rarely, we use general anesthesia.

• Incisions with a n°11 blade are made in the donor site. When possible, incisions are placed in wrinkle lines, folds, or fatty areas (abdomen flank, thigh, and knee).

Instruments for lipofilling cause minimal trauma to fatty tissue during placement.

• Cannulas vary in shape (curved or no-curved) and length (7–12 cm).

Liposuction: special blunt-tip, maximum diameter 3 mm, with small holes near the tip

○ Coleman I is used near a blood vessels or nerve. This cannula is capped on the tip with a lip that extends 180° over the distal aperture.

**45**

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results*

flat on the end to allow for dissection of the tissue.

○ Coleman V is a dissector used for scar's treatment.

**8.1 Harvesting of adipose tissue from a suitable donor site**

○ Coleman II is used in other circumstances. This cannula is not completely capped and has a lip that extends over the distal aperture about 130–150°.

○ Coleman III is used for the scars or fibrous tissue treatments. This cannula is

○ Infiltration cannula is a blunt 17G cannula with one or two distal aperture proximal to the tip: no fatty tissue should be infiltrated during the advancement of the cannula; fatty tissue is left in the pathway of the retreating blunt cannula (this method permits stable and regulated placement with minimal

Many different techniques have been proposed for harvesting of adipose tissue: the fundamental aim is minimizing adipocyte damage and increasing the survival of adipose tissue. Incisions with a n°11 blade are made in the donor site, when possible in wrinkle lines, folds, or fatty areas (abdomen, flank, thigh, and knee).

There are many different natural fat deposits: it is important an accurate preop-

The abdomen is the most common site of fat harvesting; it is also common in the trochanteric region (saddlebags) and in the medial/internal part of the thighs and knees.

In 1993, Klein proposed a new method called tumescent technique in which a fluid solution (Klein's solution) was injected into the donor site improving the safety of large-volume liposuction (it eliminated the need for general anesthesia

the use of a water-jet system to harvest the fat tissue and collect it in a closed container: minimal bruising and postoperative pain, faster harvesting time, and

into fatty tissue engorged with tumescent fluid (Klein's solution)

Another technique is the "Berlin autologous lipotransplantation", which involves

In the course of fat harvesting, a blunt cannula is inserted through an incision

• Negative pressure liposuction is faster than 10 ml syringe aspiration (low pressure) and is an effective method for aspiration of large amounts of fat, but it causes massive destruction of adipocytes, greatly reducing the survival of fat graft.

*DOI: http://dx.doi.org/10.5772/intechopen.82566*

Transplantation: small blunt-tip

**8. Surgical technique**

surgical excision.

*8.1.1 DRY technique*

*8.1.2 WET technique*

greater sterility.

erative examination of the patient.

and reduced surgical hemorrhage).

irregularities or clumps of tissue).

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results DOI: http://dx.doi.org/10.5772/intechopen.82566*

Transplantation: small blunt-tip

○ Infiltration cannula is a blunt 17G cannula with one or two distal aperture proximal to the tip: no fatty tissue should be infiltrated during the advancement of the cannula; fatty tissue is left in the pathway of the retreating blunt cannula (this method permits stable and regulated placement with minimal irregularities or clumps of tissue).

#### **8. Surgical technique**

*Osteoarthritis Biomarkers and Treatments*

The MSCs embedded into these clusters have high vitality and can still differentiate into the three mesodermal lineages as a proof of their multipotency. To prove that quality is superior to quantity, it has been demonstrated in an animal model of criti-

During the First World War, the fat was used to heal soldier wounds and its application was very heterogeneous until 1990 where Sidney Coleman defined precise guidelines. The evolution of surgical techniques associated the volumizing effect of lipofilling to the regenerative properties of natural adipose tissue, favoring the processing, the reduction, and the purification of the tissue to raise the survival of grafts and magnify the regenerative potential of MSCs. Also reducing the volume of grafts and the diameter of injection needles had helped the hosting tissue to decrease stress and trauma, thus provoking less inflammation and ameliorating again graft survival [58]. Adipose tissue is now used with lipofilling techniques in hard-toheal wounds and in all cases characterized by mortified tissue (burn, compression, and radiation) linking the regenerative potential also to an esthetic effect. Wound healing professionals use lipofilling to treat ulcers resistant to classical therapies and advanced dressing and also for the treatment of critical limb ischemia and diabetic patients [59]. In orthopedics, intra-articular lipofilling has become a fashionable and innovative strategy to fight osteoarthritis, thanks to the ability of this tissue product to reequilibrate the articular homeostasis and to reduce the inflammation of the synovial membrane [60]. The integrity of the extracellular matrix can also exploit the shock absorber function of adipose tissue reducing the stress between cartilage surfaces. The paracrine effect of MSCs can also promote cartilage repair when mechanical conditions of the articulation are stable. The anti-adhesive properties of adipose tissue were exploited in the surgery of tendons and nerves to limit the formation of fibrotic and scarring tissue, thus also limiting the relapse incidence. Finally, adipose tissue was also studied in pain management for the intradiscal infiltration for

ADSC)

isolated ADSC [57].

cal limb ischemia that an injection of 500 μl of FAT (which contain 2 × 104

restores limb perfusion better than a single injection of 1 × 106

the treatment of low back pain associated to black disc.

The technique of liposculture is originally created for esthetic purposes. Autologous fat transfer has recently become an increasingly popular surgical procedure: harvesting, refinement/processing, and transfer of subcutaneous tissue to provide relatively pure and intact parcels of fat are paramount for successful lipofilling. Usually, we use local anesthesia with sedation or epidural anesthesia.

• Incisions with a n°11 blade are made in the donor site. When possible, incisions are placed in wrinkle lines, folds, or fatty areas (abdomen flank, thigh, and

Instruments for lipofilling cause minimal trauma to fatty tissue during placement.

Liposuction: special blunt-tip, maximum diameter 3 mm, with small holes near

○ Coleman I is used near a blood vessels or nerve. This cannula is capped on the

• Cannulas vary in shape (curved or no-curved) and length (7–12 cm).

tip with a lip that extends 180° over the distal aperture.

**44**

the tip

**7. Lipofilling**

knee).

Rarely, we use general anesthesia.

#### **8.1 Harvesting of adipose tissue from a suitable donor site**

Many different techniques have been proposed for harvesting of adipose tissue: the fundamental aim is minimizing adipocyte damage and increasing the survival of adipose tissue. Incisions with a n°11 blade are made in the donor site, when possible in wrinkle lines, folds, or fatty areas (abdomen, flank, thigh, and knee).

There are many different natural fat deposits: it is important an accurate preoperative examination of the patient.

The abdomen is the most common site of fat harvesting; it is also common in the trochanteric region (saddlebags) and in the medial/internal part of the thighs and knees.

The main techniques for fat harvesting are vacuum suction, syringe suction, or surgical excision.

#### *8.1.1 DRY technique*

Other surgeons advocate a "dry" fat harvesting: cell viability has been similar to "wet" fat harvesting nut, and this technique may lead to a greater requirement for analgesic.

#### *8.1.2 WET technique*

In 1993, Klein proposed a new method called tumescent technique in which a fluid solution (Klein's solution) was injected into the donor site improving the safety of large-volume liposuction (it eliminated the need for general anesthesia and reduced surgical hemorrhage).

Another technique is the "Berlin autologous lipotransplantation", which involves the use of a water-jet system to harvest the fat tissue and collect it in a closed container: minimal bruising and postoperative pain, faster harvesting time, and greater sterility.

In the course of fat harvesting, a blunt cannula is inserted through an incision into fatty tissue engorged with tumescent fluid (Klein's solution)

• Negative pressure liposuction is faster than 10 ml syringe aspiration (low pressure) and is an effective method for aspiration of large amounts of fat, but it causes massive destruction of adipocytes, greatly reducing the survival of fat graft.

• Conventional liposuction with high negative pressure may cause disruption of 90% of adipocytes structures.

Cannula size may also impact the viability of harvested adipocytes. Performing biopsy and lipoaspiration with large-bore cannulas could reduce the risk of cellular rupture by preserving native tissue structure.

So the size of cannula must be large enough to preserve adipocytes and stromal cells and their anatomical relationship without limiting diffusion of nutrients.

Coleman described a technique for fat harvest that minimized trauma to the adipocytes.

He used 3-mm incisions (n°11 blade), a 3-mm blunt edge, 2 hole harvesting cannulas (3 mm) connected to a 10-ml Luer-Lock syringe. The cannula is pushed through the harvest site (abdomen, flank, thigh, knee, or other sites with excess adipose tissue) as the surgeon uses digital manipulation to create a gentle negative pressure.

A combination of lower negative pressure and the curetting action of the cannula through the tissues allows parcels of fat to move into the syringe. At the end, the syringe is disconnected from the cannula and replaced with a plug that seals the Luer-Lock end of the syringe.

#### **8.2 Processing**

The most commonly used methods to process fat tissue are centrifugation, sedimentation, washing, and filtration.

The goal of fat processing is:

To eliminate contaminants that can cause inflammation at the recipient site, which can be detrimental for the fat graft. These elements include cellular debris, free oil, and other nonviable components of the lipoaspirate such as hematogenous cells.

#### **8.3 Ensuring graft viability**

Centrifugation is the most widely used technique for postharvest fat processing and has previously been considered the criterion standard. Coleman's technique consisted in centrifugation (3000 rpm for 3 minutes) to separate the different components as follows:

**47**

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results*

The supernatant fat and the lower aqueous layers are discarded, leaving concen-

That is an optimal method to obtain the highest concentration of stem cells and increased angiogenic grow factors (FGF and VEGF). It separates adipocytes from blood cells and enzymes (lipids, proteases, and lipases). Sedimentation allows

Washing methods has the goal of removing superfluous tumescent fluid and all

• Lipocell technique is a new procedure that allows eliminating blood, oil, cellular debris, or other nonviable components and obtaining a pure lipoaspirate.  With a skin incision like a diameter of the cannula, the graft is put at the level of the anatomical region involved. Cannulas with small gauge will reduce tissue trauma, bleeding, and hematoma.

Through multiple access sites, multiple tunnels are created on insertion, but fat

In our experience, mainly concentrated on the knee involves the constant association of a diagnostic and surgical arthroscopy; in fact, in the majority of cases, there are meniscal lesions (flap type) or unstable chondral lesions which, if left

All steps in surgical technique (harvesting, processing, and transplantation) are important. Complications are few, rare, and minimal.

Graft can survive up to 48 hours by tissue fluid absorption.

untreated, could lead to failure of the grafting procedure alone.

larities, cellulitis, and damage to the underlying structures.

is injected only during withdrawal of the cannula.

Early identification of local sepsis.

• Pure graft filtration is a new technology that uses a closed-membrane filtration system for preparation and isolation of stromal vascular fraction and its

*DOI: http://dx.doi.org/10.5772/intechopen.82566*

obtaining a large number of vital intact adipocytes [61].

elements that can be detrimental for the fat graft.

number of adipocytes.

**8.4 Implantation/fat injection**

increas fat survival.

**9. Complications**

as fast as possible.

trated viable fat cells.

That is an optimal method to obtain the highest concentration of stem cells and increased angiogenic grow factors (FGF and VEGF). It separates adipocytes from blood cells and enzymes (lipids, proteases, and lipases). Sedimentation allows obtaining a large number of vital intact adipocytes [61].

Washing methods has the goal of removing superfluous tumescent fluid and all elements that can be detrimental for the fat graft.

• Lipocell technique is a new procedure that allows eliminating blood, oil, cellular debris, or other nonviable components and obtaining a pure lipoaspirate. This technique preserves a large number of mesenchymal stem cells and a large number of adipocytes.

Filtration allows elimination of contaminants by maintaining viable adipocytes and a lot of ADSC, thus obtaining a viable graft material for large volume fat transfers.

• Pure graft filtration is a new technology that uses a closed-membrane filtration system for preparation and isolation of stromal vascular fraction and its mechanism is known to work by principles similar to a dialysis unit.

#### **8.4 Implantation/fat injection**

*Osteoarthritis Biomarkers and Treatments*

adipocytes.

pressure.

**8.2 Processing**

fat retention.

fat.

cells.

Luer-Lock end of the syringe.

The goal of fat processing is:

confused with volume correction.

**8.3 Ensuring graft viability**

components as follows:

• Middle: primarily fatty tissue.

ing the number of ADSC in the graft material.

• Upper level: least dense and consists primarily of oil.

• Lowest: blood, water, and aqueous elements.

90% of adipocytes structures.

rupture by preserving native tissue structure.

• Conventional liposuction with high negative pressure may cause disruption of

Cannula size may also impact the viability of harvested adipocytes. Performing biopsy and lipoaspiration with large-bore cannulas could reduce the risk of cellular

So the size of cannula must be large enough to preserve adipocytes and stromal

cells and their anatomical relationship without limiting diffusion of nutrients.  The cannula is pushed through the harvest site (abdomen, flank, thigh, knee, or other sites with excess adipose tissue) as the surgeon uses digital manipulation to create a gentle negative

A combination of lower negative pressure and the curetting action of the cannula through the tissues allows parcels of fat to move into the syringe. At the end, the syringe is disconnected from the cannula and replaced with a plug that seals the

The most commonly used methods to process fat tissue are centrifugation, sedimentation, washing, and filtration. Comparative studies investigating the effects of fat processing with different techniques have showed no significant differences in

To eliminate contaminants that can cause inflammation at the recipient site, which can be detrimental for the fat graft. These elements include cellular debris, free oil, and other nonviable components of the lipoaspirate such as hematogenous

• Blood must be extracted in order to improve degradation of the transplanted

• Moreover, many authors report an improvement in graft viability by maximiz-

Centrifugation is the most widely used technique for postharvest fat processing and has previously been considered the criterion standard. Coleman's technique consisted in centrifugation (3000 rpm for 3 minutes) to separate the different

• Since the debris will be absorbed after a few hours, its injection could be

**46**

Principles of fat reimplantation are found on optimal recipient site vascularity to increas fat survival.

Graft can survive up to 48 hours by tissue fluid absorption.

Neovascularization progresses 1 mm/day; therefore, a deposit diameter greater than 2 mm should be avoided to prevent central necrosis. With a skin incision like a diameter of the cannula, the graft is put at the level of the anatomical region involved. Cannulas with small gauge will reduce tissue trauma, bleeding, and hematoma.

Through multiple access sites, multiple tunnels are created on insertion, but fat is injected only during withdrawal of the cannula.

In our experience, mainly concentrated on the knee involves the constant association of a diagnostic and surgical arthroscopy; in fact, in the majority of cases, there are meniscal lesions (flap type) or unstable chondral lesions which, if left untreated, could lead to failure of the grafting procedure alone.

#### **9. Complications**

All steps in surgical technique (harvesting, processing, and transplantation) are important. Complications are few, rare, and minimal.  The chances of survival are higher if the fat graft is less manipulated and reinjected as fast as possible.

Early identification of local sepsis.

### **10. Conclusion**

In conclusion, there are several treatments for knee OA including nonpharmacological and pharmacological treatments.

Among the nonpharmacological ones, patient education and self-management strategies, advising weight loss and strengthening programs are included.

Regarding the pharmacological treatment, the NSAIDs can be used in the shortterm therapy but their effect is limited in time.

Another employed strategy to manage knee OA is represented by joint injections of corticosteroids, hyaluronic acid, PRP, and even stem cells [47].

The reason for using ASCs in orthopedics is derived from tissue engineering studies that enhance their ability to differentiate into osteoblastic or chondrocyte using appropriate culture media and bioengineered structures that can accommodate cells as a biological scaffold.

The studies of Hattori and colleagues showed an osteogenic differentiation (by electron microscope, with histological evaluation and by the capacity of osteocalcin secretion) analogous to BMSC using a beta-tricalciophosphate scaffold [62, 63].

Always Hattori and colleagues in 2008 proposed new strategies for the in vitro expansion of ASCs. In most cases, the expansion was obtained with fetal bovine serum (FBS) which, in subsequent clinical applications, could have caused infections or immunological reactions caused by the proteins present in the FBS [64].

For this reason, Hattori and colleagues have demonstrated, with studies on the mouse, that it is possible to obtain the expansion of the ASC with the same differentiation potentials, using a small amount of autologous serum containing type I, FGF-2 collagen and thus opening the way to possible therapeutic applications.

Promising is the application in the treatment of cartilaginous lesions. In 2007, Masuoka and colleagues used a three-dimensional honeycomb scaffold of atelocollagene (ACHMS scaffold) with ASCs for cartilaginous lesions in rabbit's knees; as a control group, they used the only ACHMS-scaffold or nothing [65]. Twelve weeks later, histological analyzes have highlighted that only in cases where ASC + ACHMS-scaffold had been used, there was hyaline cartilage with high expression of type II collagen. It should be also noted that the ASCs, as mentioned above, have the ability to release factors of tissue growth and/or regeneration and cytokines. These substances play an important role in chemotaxis, in promoting tissue regeneration, cell differentiation, and neoangiogenesis.

Regenerative medicine opens the way for a new therapeutic frontier, as it now allows an improvement in symptoms and in the functionality of the joints. On the other hand, further studies are needed, major follow-ups, targeted clinical trials, and finally the possibility of a second look to evaluate and validate the real regenerative capacity of this treatment.

**49**

**Author details**

Edoardo De Fenu1

Andrea Conversi1

provided the original work is properly cited.

2 Mater Dei Sport Clinique, Rome, Italy

3 Mater Dei Clinique, Rome, Italy

\*, Berardino Di Paola<sup>2</sup>

and Ezio Adriani2

1 Policlinico Umberto I Università Sapienza, Rome, Italy

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 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,

, Marco Ruggiero3

, Bruno Carlesimo3

,

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results*

*DOI: http://dx.doi.org/10.5772/intechopen.82566*

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results DOI: http://dx.doi.org/10.5772/intechopen.82566*

### **Author details**

*Osteoarthritis Biomarkers and Treatments*

logical and pharmacological treatments.

date cells as a biological scaffold.

term therapy but their effect is limited in time.

In conclusion, there are several treatments for knee OA including nonpharmaco-

Among the nonpharmacological ones, patient education and self-management

Regarding the pharmacological treatment, the NSAIDs can be used in the short-

Another employed strategy to manage knee OA is represented by joint injections

The reason for using ASCs in orthopedics is derived from tissue engineering studies that enhance their ability to differentiate into osteoblastic or chondrocyte using appropriate culture media and bioengineered structures that can accommo-

The studies of Hattori and colleagues showed an osteogenic differentiation (by electron microscope, with histological evaluation and by the capacity of osteocalcin secretion) analogous to BMSC using a beta-tricalciophosphate scaf-

Always Hattori and colleagues in 2008 proposed new strategies for the in vitro expansion of ASCs. In most cases, the expansion was obtained with fetal bovine serum (FBS) which, in subsequent clinical applications, could have caused infections or immunological reactions caused by the proteins present in

For this reason, Hattori and colleagues have demonstrated, with studies on the mouse, that it is possible to obtain the expansion of the ASC with the same differentiation potentials, using a small amount of autologous serum containing type I, FGF-2 collagen and thus opening the way to possible therapeutic

Promising is the application in the treatment of cartilaginous lesions. In 2007, Masuoka and colleagues used a three-dimensional honeycomb scaffold of atelocollagene (ACHMS scaffold) with ASCs for cartilaginous lesions in rabbit's knees; as a control group, they used the only ACHMS-scaffold or nothing [65]. Twelve weeks later, histological analyzes have highlighted that only in cases where ASC + ACHMS-scaffold had been used, there was hyaline cartilage with high expression of type II collagen. It should be also noted that the ASCs, as mentioned above, have the ability to release factors of tissue growth and/or regeneration and cytokines. These substances play an important role in chemotaxis, in promoting tissue regeneration, cell differentiation, and

Regenerative medicine opens the way for a new therapeutic frontier, as it now allows an improvement in symptoms and in the functionality of the joints. On the other hand, further studies are needed, major follow-ups, targeted clinical trials, and finally the possibility of a second look to evaluate and validate the real regen-

strategies, advising weight loss and strengthening programs are included.

of corticosteroids, hyaluronic acid, PRP, and even stem cells [47].

**10. Conclusion**

fold [62, 63].

the FBS [64].

applications.

neoangiogenesis.

erative capacity of this treatment.

**48**

Edoardo De Fenu1 \*, Berardino Di Paola<sup>2</sup> , Marco Ruggiero3 , Bruno Carlesimo3 , Andrea Conversi1 and Ezio Adriani2

1 Policlinico Umberto I Università Sapienza, Rome, Italy

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 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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Physical Medicine and Rehabilitation. 2012;**4**(Suppl 5):S10-S19

[17] De Filippis L, Gulli S, Caliri A, et al. Epidemiology and risk factors in osteoarthritis: Literature review data from "OASIS" study. Reumatismo. 2004;**56**(3):169-184

[18] Lavernia CJ, Sierra RJ, Grieco FR. Osteonecrosis of the femoral head. The Journal of the American Academy of Orthopaedic Surgeons. 1999;**7**(4):250-261

[19] Urbaniak JR, Jones JP Jr, editors. Osteonecrosis: Etiology, Diagnosis, and Treatment. Rosemont, IL: American Academy of Orthopaedic Surgeons; 1997

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[21] Hinton R, Moody RL, Davis AW, et al. Osteoarthritis: Diagnosis and therapeutic considerations. American Family Physician. 2002;**65**(5):841-849

[22] Pool RR. Pathologic manifestations of joint disease in the athletic horse. In: McIlwraith CW, Trotter GW, editors. Joint Disease in the Horse. Philadelphia: WB Saunders; 1996. pp. 87-104

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[24] Lozada C. Osteoarthritis in Medscape reference. 2012. Available at: http://emedicine.medscape.com/ article/330487-overview

[25] Prockop DJ. Heritable osteoarthritis: Diagnosis and possible modes of cell and gene therapy. Osteoarthritis and Cartilage. 1994;**7**(4):364-366

[26] Fortier LA, Nixon AJ, Mohammed HO, et al. Altered biological activity of equine chondrocytes cultured in a three-dimensional fibrin matrix and supplemented with transforming growth factor beta-1. American Journal of Veterinary Research. 1997;**58**(1):66-70

[27] Iqbal J, Dudhia J, Bird JL, et al. Age-related effects of TGF-b on proteoglycan synthesis in equine articular cartilage. Biochemical and Biophysical Research Communications. 2000;**274**(2):467-471

[28] Frisbie DD, Sandler EA, Trotter GW, et al. Metabolic and fitogeni activities of insulin-like growth factor-1 in interleukin-1-conditioned equine cartilage. American Journal of Veterinary Research. 2000;**61**(4):436-441

[29] Tung JT, Arnold CE, Alexander LH, et al. Evaluation of the influence of prostaglandin E2 on recombinant equine interleukin-1b-stimulated matrix metalloproteinases 1, 3, and 13 and tissue inhibitor of matrix metalloproteinase 1 expression in equine chondrocyte cultures. American Journal of Veterinary Research. 2002;**63**(7):987-993

[30] Burrage PS, Mix KS, Brinckerhoff CE. Matrix metalloproteinases: Role in arthritis. Frontiers in Bioscience. 2006;**11**:529-543

[31] Brama PA, van den Boom R, DeGroott J, et al. PR Collagenase-1 (MMP-1) activity in equine synovial fluid: Influence of age, joint pathology, exercise and repeated arthrocentesis. Equine Veterinary Journal. 2004;**36**(1):34-40

[32] Platt D. Articular cartilage homeostasis and the role of growth factors and cytokines in regulating matrix composition. In: McIlwraith CW, Trotter GW, editors. Joint Disease in

**50**

*Osteoarthritis Biomarkers and Treatments*

[1] Goodman S. Osteoarthritis. In: Yee A, Paget S, editors. Expert Guide to Rheumatology. Philadelphia, PA: American College of Physicians; 2005. [9] Zhang W, Moskowitz RW, Nuki G, Abramson S, Altman RD, Arden N, et al. OARSI recommendations for the management of hip and knee osteoarthritis, Part II: OARSI evidence-based, expert consensus guidelines. Osteoarthritis and Cartilage.

[10] American Academy of Orthopaedic

Surgeons Board of Directors. Treatment of Osteoarthritis of the Knee Evidence-based Guideline. 2nd ed. 2013. Journal of the American Academy of Orthopaedic Surgeons. Sep

[11] Centers for Disease Control and Prevention. Prevalence of doctor-diagnosed arthritis and possible arthritis—30 states, 2002. MMWR. Morbidity and Mortality Weekly Report. 2004;**53**:383-385

[12] Bitton R. The economic burden of osteoarthritis. The American Journal of Managed Care. 2009;**15**(Suppl 8):

[13] Murphy L, Cisternas M, Yelin E, et al. Update: Direct and indirect costs of arthritis and other rheumatic conditions—United States, 1997. MMWR. Morbidity and Mortality Weekly Report. 2004;**53**(18):388-389

[14] Murphy L, Schwartz TA, Helmick CG, et al. Lifetime risk of symptomatic knee osteoarthritis. Arthritis and Rheumatism. 2008;**59**:1207-1213

[15] Culliford DJ, Maskell J, Kiran A, et al. The lifetime risk of total hip and knee arthroplasty: Results from the UK general practice research database. Osteoarthritis and Cartilage.

[16] Suri P, Morgenroth DC, Hunter DJ. Epidemiology of osteoarthritis and associated comorbidities.

2012;**20**:519-524

2008;**16**:137-162

2013;**21**(9):571-576

S230-S235

[2] Bijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: An update with relevance for clinical practice. Lancet.

[3] DiCesare PE, Abramson S, Samuels J. Pathogenesis of osteoarthritis. In: Firestein GS, Kelley WN, editors. Kelley's Textbook of Rheumatology. 8th ed. Philadelphia, PA, Saunders Elsevier;

[4] Bateman JF. Genetic aspects of osteoarthritis. Seminars in Arthritis and Rheumatism. 2005;**34**(6 Suppl 2):15-18

[5] Ryder JJ, Garrison K, Song F, et al. Genetic associations in peripheral joint osteoarthritis and spinal degenerative

disease: A systematic review. Annals of the Rheumatic Diseases.

[6] Valdes AM, Spector TD. The genetic epidemiology of osteoarthritis. Current Opinion in Rheumatology.

[7] McAlindon TE, Bannuru RR, Sullivan MC, Arden NK, Berenbaum F, Bierma-Zeinstra SM, et al. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthritis and Cartilage.

[8] Smink AJ, van den Ende CH, Vliet Vlieland TP, Swierstra BA, Kortland JH, Bijlsma JW, et al. "Beating osteoARThritis": Development of a stepped care strategy to optimize utilization and timing of non-surgical treatment modalities for patients with hip or knee osteoarthritis. Clinical Rheumatology. 2011;**30**:1623-1629

2008;**67**(5):584-591

2010;**22**(2):139-143

2014;**22**:363-388

**References**

pp. 269-283

2009

2011;**377**:2115-2126

the Horse. Philadelphia: WB Saunders; 1996. pp. 29-40

[33] Loeser RF. Systemic and local regulation of articular cartilage metabolism: Where does leptin fit in the puzzle? Arthritis and Rheumatism. 2003;**48**(11):3009-3012

[34] Jordan KM, Arden NK, Doherty M, Bannwarth B, Bijlsma JWJ, Dieppe P, et al. EULAR recommendations 2003: An evidence based medicine approach to the management of knee osteoarthritis. Report of a task force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT). Annals of the Rheumatic Diseases. 2003;**62**:1145-1155

[35] Punzi L, Canesi B, Carrabba M, Cimmino MA, Frizziero L, Lapadula G, et al. Consensus italiana sulle raccomandazioni EULAR 2003 per il trattamento della gonartrosi. Reumatismo. 2004;**56**:190-201

[36] Bert JM, Bert TM. Nonoperative treatment of unicompartmental arthritis: From bracing to injection. Clinics in Sports Medicine. 2014;**33**(1):1-10

[37] Uthman I, Raynauld JP, Haraoui B. Intra-articular therapy in osteoarthritis. Postgraduate Medical Journal. 2003;**79**(934):449-453

[38] Douglas RJ. Corticosteroid injection into the osteoarthritic knee: Drug selection, dose, and injection frequency. International Journal of Clinical Practice. 2012;**66**(7):699-704

[39] McGarry JG, Daruwalla ZJ. The efficacy, accuracy and complications of corticosteroid injections of the knee joint. Knee Surgery, Sports Traumatology, Arthroscopy. 2011;**19**(10):1649-1654

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[41] HK V, Percival SS, Conrad BP, Seay AN, Montero C, Vincent KR. Hyaluronic acid (HA) viscosupplementation on synovial fluid inflammation in knee osteoarthritis: A pilot study. The Open Orthopaedics Journal. 2013;**7**:378-384

[42] Mazzucco L, Balbo V, Cattana E, Guaschino R, Borzini P. Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRPKit, Plateltex and one manual procedure. Vox Sanguinis. 2009;**97**(02):110-118

[43] Molloy T, Wang Y, Murrell G. The roles of growth factors in tendon and ligament healing. Sports Medicine. 2003;**33**(05):381-394

[44] Staudenmaier R, Froelich K, Birner M, et al. Optimization of platelet isolation and extraction of autogenous TGF-beta in cartilage tissue engineering. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology. 2009;**37**(06):265-272

[45] Paterson KL, Nicholls M, Bennell KL, Bates D. Intra-articular injection of photo-activated platelet-rich plasma in patients with knee osteoarthritis: A double-blind, randomized controlled pilot study. BMC Musculoskeletal Disorders. 2016;**17**(01):67. DOI: 10.1186/ s12891-016-0920-3

[46] Kavadar G, Demircioglu DT, Celik MY, Emre TY. Effectiveness of platelet-rich plasma in the treatment of moderate knee osteoarthritis: A randomized prospective study. Journal of Physical Therapy Science. 2015;**27**(12):3863-3867

[47] Smith PA. Intra-articular autologous conditioned plasma injections

**53**

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*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results*

[54] Chen L, Tredget EE, Wu PYG, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008;**3**(4):e1886. https://doi. org/10.1371/journal.pone.0001886

[55] Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cells

International. 2012;**2012**:812693. DOI: 10.1155/2012/812693. Epub 2012 Apr 12

[57] Bianchi F, Olivi E, Baldassarre M, Giannone FA, Laggetta M, Valente S, et al. Lipogems, a new modality of fat tissue handling to enhance tissue repair in chronic hind limb ischemia. CellR4.

[58] Tremolada C, Palmieri G, Ricordi C. Adipocyte transplantation and stem cells: Plastic surgery meets regenerative

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*DOI: http://dx.doi.org/10.5772/intechopen.82566*

provide safe and efficacious treatment for knee osteoarthritis: An FDA-

sanctioned, randomized, double-blind, placebocontrolled clinical trial. The American Journal of Sports Medicine.

[48] Moraes VY, Lenza M, Tamaoki MJ, Faloppa F, Belloti JC. Plateletrich therapies for musculoskeletal soft tissue injuries. Cochrane Database of Systematic Reviews. 2014;**4**(04):CD010071. DOI: 10.1002/14651858.CD010071.pub2

[49] Grambart ST. Sports medicine and platelet-rich plasma: Nonsurgical therapy. Clinics in Podiatric Medicine and Surgery. 2015;**32**(01):99-107

[50] Duymus TM, Mutlu S, Dernek B, Komur B, Aydogmus S, Kesiktas FN. Choice of intra-articular injection in treatment of knee osteoarthritis: Platelet-rich plasma, hyaluronic acid or ozone options. Knee Surgery, Sports Traumatology, Arthroscopy.

[51] DeChellis DM, Cortazzo MH. Regenerative medicine in the field of pain medicine: Prolotherapy, platelet-rich plasma therapy, and stem cell therapy—Theory and evidence. Techniques in Regional Anesthesia and Pain Management. 2011;**15**(02):74-80

[52] Murphy MB, Moncivais K, Caplan AI. Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Experimental & Molecular Medicine. 2013;**45**:e54. DOI: 10.1038/emm.2013.94

[53] Doorn J, van de Peppel J, van Leeuwen JP, Groen N, van Blitterswijk CA, de Boer J. Proosteogenic trophic effects by PKA activation in human mesenchymal stromal cells. Biomaterials. Sep, 2011;**32**(26):6089-6098. DOI: 10.1016/j. biomaterials.2011.05.010. Epub 2011

2017;**25**(02):485-492

2016;**44**(04):884-891

*Fat Tissue's Graft in Osteoarthritis Treatment: Indications, Preparations, and Results DOI: http://dx.doi.org/10.5772/intechopen.82566*

provide safe and efficacious treatment for knee osteoarthritis: An FDAsanctioned, randomized, double-blind, placebocontrolled clinical trial. The American Journal of Sports Medicine. 2016;**44**(04):884-891

*Osteoarthritis Biomarkers and Treatments*

[33] Loeser RF. Systemic and local regulation of articular cartilage metabolism: Where does leptin fit in the puzzle? Arthritis and Rheumatism.

2003;**48**(11):3009-3012

Diseases. 2003;**62**:1145-1155

[35] Punzi L, Canesi B, Carrabba M, Cimmino MA, Frizziero L, Lapadula G, et al. Consensus italiana sulle raccomandazioni EULAR 2003 per il trattamento della gonartrosi. Reumatismo. 2004;**56**:190-201

[36] Bert JM, Bert TM. Nonoperative treatment of unicompartmental arthritis: From bracing to injection.

[37] Uthman I, Raynauld JP, Haraoui

osteoarthritis. Postgraduate Medical Journal. 2003;**79**(934):449-453

International Journal of Clinical Practice. 2012;**66**(7):699-704

[39] McGarry JG, Daruwalla ZJ. The efficacy, accuracy and complications of corticosteroid injections of the knee joint. Knee Surgery, Sports Traumatology, Arthroscopy. 2011;**19**(10):1649-1654

[40] Altman RD, Manjoo A, Fierlinger A, Niazi F, Nicholls M. The mechanism

[38] Douglas RJ. Corticosteroid injection into the osteoarthritic knee: Drug selection, dose, and injection frequency.

Clinics in Sports Medicine.

B. Intra-articular therapy in

2014;**33**(1):1-10

1996. pp. 29-40

the Horse. Philadelphia: WB Saunders;

of action for hyaluronic acid treatment in the osteoarthritic knee: A systematic review. BMC Musculoskeletal Disorders.

[41] HK V, Percival SS, Conrad BP, Seay AN, Montero C, Vincent KR. Hyaluronic acid (HA) viscosupplementation on synovial fluid inflammation in knee osteoarthritis: A pilot study. The Open Orthopaedics Journal. 2013;**7**:378-384

[42] Mazzucco L, Balbo V, Cattana E, Guaschino R, Borzini P. Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRPKit, Plateltex and one manual procedure. Vox Sanguinis.

[43] Molloy T, Wang Y, Murrell G. The roles of growth factors in tendon and ligament healing. Sports Medicine.

[44] Staudenmaier R, Froelich K, Birner M, et al. Optimization of platelet isolation and extraction of autogenous TGF-beta in cartilage tissue engineering. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology.

[45] Paterson KL, Nicholls M, Bennell KL, Bates D. Intra-articular injection of photo-activated platelet-rich plasma in patients with knee osteoarthritis: A double-blind, randomized controlled pilot study. BMC Musculoskeletal Disorders. 2016;**17**(01):67. DOI: 10.1186/

[46] Kavadar G, Demircioglu DT, Celik MY, Emre TY. Effectiveness of platelet-rich plasma in the treatment of moderate knee osteoarthritis: A randomized prospective study. Journal of Physical Therapy Science.

[47] Smith PA. Intra-articular autologous

2009;**97**(02):110-118

2003;**33**(05):381-394

2009;**37**(06):265-272

s12891-016-0920-3

2015;**27**(12):3863-3867

conditioned plasma injections

2015;**16**:321

[34] Jordan KM, Arden NK, Doherty M, Bannwarth B, Bijlsma JWJ, Dieppe P, et al. EULAR recommendations 2003: An evidence based medicine approach to the management of knee osteoarthritis. Report of a task force of the Standing Committee for International Clinical Studies Including Therapeutic Trials (ESCISIT). Annals of the Rheumatic

**52**

[48] Moraes VY, Lenza M, Tamaoki MJ, Faloppa F, Belloti JC. Plateletrich therapies for musculoskeletal soft tissue injuries. Cochrane Database of Systematic Reviews. 2014;**4**(04):CD010071. DOI: 10.1002/14651858.CD010071.pub2

[49] Grambart ST. Sports medicine and platelet-rich plasma: Nonsurgical therapy. Clinics in Podiatric Medicine and Surgery. 2015;**32**(01):99-107

[50] Duymus TM, Mutlu S, Dernek B, Komur B, Aydogmus S, Kesiktas FN. Choice of intra-articular injection in treatment of knee osteoarthritis: Platelet-rich plasma, hyaluronic acid or ozone options. Knee Surgery, Sports Traumatology, Arthroscopy. 2017;**25**(02):485-492

[51] DeChellis DM, Cortazzo MH. Regenerative medicine in the field of pain medicine: Prolotherapy, platelet-rich plasma therapy, and stem cell therapy—Theory and evidence. Techniques in Regional Anesthesia and Pain Management. 2011;**15**(02):74-80

[52] Murphy MB, Moncivais K, Caplan AI. Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Experimental & Molecular Medicine. 2013;**45**:e54. DOI: 10.1038/emm.2013.94

[53] Doorn J, van de Peppel J, van Leeuwen JP, Groen N, van Blitterswijk CA, de Boer J. Proosteogenic trophic effects by PKA activation in human mesenchymal stromal cells. Biomaterials. Sep, 2011;**32**(26):6089-6098. DOI: 10.1016/j. biomaterials.2011.05.010. Epub 2011 May 31

[54] Chen L, Tredget EE, Wu PYG, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008;**3**(4):e1886. https://doi. org/10.1371/journal.pone.0001886

[55] Baer PC, Geiger H. Adipose-derived mesenchymal stromal/stem cells: tissue localization, characterization, and heterogeneity. Stem Cells International. 2012;**2012**:812693. DOI: 10.1155/2012/812693. Epub 2012 Apr 12

[56] Chen H, Lee MJ, Chen CH, Chuang SC, Chang LF, Ho ML, et al. Proliferation and differentiation potential of human adiposederived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures. Journal of Cellular and Molecular Medicine. Mar, 2012;**16**(3):582-593. DOI: 10.1111/j.1582-4934.2011.01335.x

[57] Bianchi F, Olivi E, Baldassarre M, Giannone FA, Laggetta M, Valente S, et al. Lipogems, a new modality of fat tissue handling to enhance tissue repair in chronic hind limb ischemia. CellR4. 2014;**2**(6):e1289

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Section 3

Natural Treatments for

Osteoarthritis

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Section 3


*2.3. Variable Selection*

The environmental variables (n = 98) were reduced by the variable inflation factor (*VIF* < 10) [28] and the AUCRF R package [36,37]. We considered the most parsimonious model, i.e., the one with lower number of variables [28], built with the non-collinear variables that maximized the AUC value of the Random Forest (RF) model prediction [36,37]. This index was calculated as:

$$VIF = \frac{1}{1 - R^2} \tag{1}$$

where *R*2 is the coefficient of determination. The selection of non-collinear variables was carried out using the stepwise procedure with the *usdm* package in the *R* program, depending on the importance of each variable [38] in the *R* program [39]. It was determined through simple linear correlations between the predictions of the model including all the variables (full model) and the prediction excluding the evaluated variable (reduced model) [28].

The AUCRF R package implements a stepwise variable selection procedure and returning the AUC value reached. The order in which the variables are added to the model is estimated according to the variable important measurement. The variable importance measurement offers two different importance measures, the mean decrease Gini (MDG) and mean decrease accuracy (MDA), respectively. Furthermore, the probability that the variable is elected to build the model. For more details see [36]. Hence, the model is firstly feed with the variable that returns the best AUC and new variables are added until the model is built with all available variables. The package returns the number and identity of the variables which give the best AUC value with their variable importance and probability of selection score [36].


**4.15. GLUT10** *(SLC2A10)*

GLUT8 raised the question of whether this isoform would be insulin-responsive and thereby compensate for GLUT4 in the respective knockout mice which are lacking the insulinresponsive glucose transporter yet near normal glucose tolerance. Indeed, in blastocysts of mice, GLUT8 has been found to account for insulin-stimulated glucose uptake. However, various groups performed extensive studies in primary rat adipocytes, 3T3L1 adipocytes, insulin-responsive CHO-cells as well as neuronal cell types such as N2A, PC12, and hippocampal neurons that all failed to identify a stimulus that leads to a plasma mem‐

The intracellular localization of GLUT8 is also observed *in vivo* under steady-state conditions. In the testis immunofluorescence microscopy shows intracellular localization of GLUT8 in a late-endosomal/lysosomal compartment. In the brain, immunogold labeling electron-micro‐ scopy localized GLUT8 in synaptic dense core vesicles of nerve terminals and secretory

The intracellular retention signal of GLUT8 has been characterized and found to contain the consensus sequence [D/E]XXXL[L/I] that represents a late-endosomal/lysosomal sorting motif. The DEXXXLL sorting signal of GLUT8 interacts with the adaptor proteins AP-1 and AP-2 and controls trafficking of the protein. Interestingly, both GLUT8 and GLUT12 contain a [D/ E]XXXL[L/I] sorting signal; however, the exact composition of the XXX residues seems to fine-

GLUT8 knockout mice appear healthy and exhibit normal growth, body weight development and glycemic control, indicating that GLUT8 does not play a significant role in maintenance of whole body glucose homeostasis. Offspring distribution from heterozygous matings indicated a deviation from the expected Mendelian distribution regarding birth of GLUT8 homozygous animals. This observation was attributed to a decreased sperm motility of GLUT8 deficient spermatocytes that is associated with lower ATP levels and a reduced mitochondrial membrane potential, while number and survival rate of spermatozoa is unchanged. The reduced amount of homozygous GLUT8 offspring is not related to impaired preimplantation embryo development – as might have been suggested by antisense studies in embryos-since mating of knockout mice produced viable, normally developing offspring in numbers comparable to those of a wildtype intercross. GLUT8 deficiency is associated with behavioural alterations indicating a significant physiological role for this isoform in the central nervous system. GLUT8 deficient mice have an increased proliferation of hippocampal cells and behavioral tests show increased arousal, a tendency to altered grooming, and a reduced risk assessment in those animals. However, despite the in depth characterization of the cell biology of GLUT8 and its physiology *in vivo* based on knockout mice, the function of the protein

brane translocation of GLUT8.

30 Glucose Homeostasis

granules of vasopressin neurons.

*4.14.1. GLUT8 physiology*

remains thus far unknown.

*Mouse Models of GLUT8 deficiency*

tune the routing and trafficking of the two proteins.

GLUT10 has been cloned from human liver cDNA by 3' and 5' RACE PCR based on an EST sequence that was identified via a homology search with known GLUT protein sequences. GLUT10 mRNA is present in the human heart, lung, brain, liver, skeletal muscle, pancreas, placenta and kidney. Expression of GLUT10 is also detected in human and mouse white adipose tissue as well as human and mouse adipocyte cell lines SGBS and 3T3L1, respectively. One remarkable structural feature is the absence of the PESPR motif just after helix 6 that is conserved for all the GLUT isoforms. Heterologous expression of GLUT10 in *Xenopus laevis* oocytes demonstrated 2-DG transport with high affinity (Km ~0.3 mM). Uptake of 2-DG can be competed with galactose and glucose and inhibited with phloretin.

Although no localization study has been performed for GLUT10 clearly demonstrating a plasma membrane or intracellular localization as seen for other Class III family members, immunocytochemical studies indicated an intracellular localization for GLUT10 under steadystate conditions. The presence of the potential internalization motif YSRI at the C terminus of the transporter supports these findings.

GLUT10 is located at the chromosomal region 20q12-13.1, a susceptibility locus that has been linked to type 2 diabetes in Caucasian Americans. Therefore, there has been particular interest in GLUT10 as a potential candidate or susceptibility gene involved in the disease. However, polymorphisms in GLUT10 were not associated to type 2 diabetes in Caucasian Americans, Danish, Finns, and Taiwanese populations.

## *4.15.1. GLUT10 physiology*


**4. Insulin resistance**

Patients with T2DM have high whole body glucose production and glucogenesis but low glycogenolysis compared with normal subjects. In patients with IR, there is impairment of glucose transport and insulin signaling in target tissues with release of inflammatory markers from the adipose tissue [23, 24]. The glucose, FFA, autonomic nerves, fat-derived hormones and the gut hormone glucagon-like peptide-1 (GLP-1) are mediators sending signals to the βcells to respond to IR. The maintenance of normal glucose and lipid metabolism is by the reciprocal relation of IR to IS and insulin secretion. Hyperbolic relation is the best description of the curvilinear relation between IS and secretion. This relation is impaired with failure of these signals to act on β-cells to secret insulin ends with subsequent development of dysgly‐ cemia (Impaired Fasting glucose- IFG, impaired Glucose tolerance –IGT and DM) [25].

Obesity is the most common cause of IR and T2DM. Simply being overweight (BMI >25) raises the risk of developing T2DM by a factor of 3 [26].

The main components of IR are dysglycemia, dyslipidemia, obesity, hypertension and hyperinsulinemia. Therefore, it is the key feature of metabolic syndrome (MS) and vascular complications (cardiovascular and stroke). IR components once are acquired, those with genetic predisposition will develop the full picture of the disorder suggesting the final phenotypic expression involves both genetic and acquired influences. The most important environmental factor in IR is central obesity which is mainly caused by intake of high fat, and refined carbohydrate without physical activity. These are exacerbated by genetic predisposi‐ tion but IR could be reduced with minimizing dietary intake and regular exercise [27].

The three potential mechanisms of the controlling glucose metabolism in the skeletal muscles are the glycogen synthase, the hexokinase and the major insulin-stimulated glucose trans‐ porter GLUT4. Therefore, defects in glycogen synthesis in the skeletal muscles playing a major role in the pathogenesis of IR [28]. The decrease in the ability of normal responding skeletal muscles to circulating insulin levels or concentrations is main principle of development of IR which could precede the overt T2DM by 10 to 20years [28].

T2DM is characterized by increased hepatic glucose output, increased peripheral resistance to insulin action (due to receptor and postreceptor defects) and impaired insulin secretion.

Two major variants of insulin receptor abnormalities associated with acanthosis nigricans, hyperinsulinemia and marked hyperandrogenism. The classic type A IR syndrome, which is due to genetic defect in the insulin-signaling system such as mutation in the insulin receptor gene [29] and type B IR syndrome, which results from autoantibodies to the insulin receptor [30].

Many factors could enhance IR include, obesity, inflammation and inflammatory markers, defects in genes and drugs. These will be demonstrated further in this chapter.


**4.3 Business model for knowledge capitalization**

The middle managers normally follow up on the statistics on a monthly basis. This could, however, in some situations be changed. Especially, if changes have been made, follow-ups could be done more often. The departments follow up on both a daily and a weekly basis, especially the number of discharges. Three different IT systems are used to follow up. In the case of warnings, or if the staff recognizes bottlenecks, analyses of what is happening and follow-ups will be conducted. Of course, all follow-ups result in some learning, but the knowledge is not diffused and implemented in the organization in a predefined way. However, the analyses further guide how the capacity is dimensioned. People at all levels, from the political and management levels, to the staff level at the hospital, who all naturally want to contribute to developing and improving the health care, get support from the data that the IT systems provide. Especially, the internal work is much in focus, to avoid queuing of patients.

total budget for production of health care at each hospital. This dimensioning then affects whether patients need to be rejected or referred to another health-care provider, maybe to another health-care level, such as a primary health-care center.

For each of the three themes, the key results are presented in **Table 1**.

actions

*Operations Knowledge Management in Health Care DOI: http://dx.doi.org/10.5772/intechopen.93793*

resources

resources

of disease

• Desire to have performance data in real time to learn and take appropriate

• A fixed number of operating rooms based on availability determines the

• Coordinators from different departments meet to exchange knowledge

• Desire to have knowledge to match the staff schedule with the needs

• Many different IT systems are used in parallel and in different ways

• A fixed set of standardized test packages is sometimes used for each group

• Oral analyses at departments on a daily basis to learn and create knowledge

• Number of admissions in the care agreement, but each admission can vary

• Collaboration with other health-care providers is needed for frail elderly

• Professionals share knowledge about the status of patients at daily

• Brainstorming for improvements is conducted at department meetings

• Middle managers follow up on the statistics at least on a monthly basis • Department managers follow up on a daily and weekly basis • In the case of warnings, follow-ups and analyses are conducted • Analyses guide the dimensioning of capacity, especially to avoid queuing

• The number of admissions says very little about the resources needed • Unclear how to estimate resource use: based on number of admissions or

• One goal is to decrease the number of visits at the emergency department • Politicians use statistics to allocate money to different health-care areas

• Planning and following up part of the daily management • Ad hoc-based recognitions of changes can cause a reassessment of

• Historical numbers are used for the emergency department

• Knowledge of shortcomings could save a lot of resources

• No formally structured learning and knowledge creation

• Excel sheets are used for scheduling and planning • Manual routines that the staff have to be reminded of • Difficult to rely on manually registered data • Dimensioning of resources is needed for planned visits • Scheduling needs to be done by experienced staff

• Monitoring of inflow and outflow of in-patients

• An ordered amount of health care should be produced

• The total number of patients is decided at the regional level • Departments use statistics to improve their activities and processes

• Knowledge is not diffused in a predefined way

• Follow-ups on an occasionally basis • Analyses and follow-ups are not documented • No routines for documenting work performance

in number of days

patients

meetings

of patients

*The three themes analyzed in the study and the results.*

average admission time

• Staff adapts to changes in admission of patients

• A need to manage required resources better

about inflow and outflow

**4.4 Summary of results**

Learning and knowledge creation

Organizational architecture for adaptive and exaptive

Business model for knowledge capitalization

**Table 1.**

**261**

capacity

culture

The regional administration orders the amount of health care that should be produced at the hospital. The provision of health care is measured by the number of admission sessions rather than the number of admission days. This indicator does not say much about the resources needed and used, because each admission session can vary considerably in length. This indicator also makes it very difficult for the hospital managers to follow up on the situation, and it provides little information that is relevant for the dimensioning of resources and capacity. To make sure the admitted patients receive effective health care and improve the economic figures, it is important to plan the discharge already when the patients are admitted. Implicitly, this means that the hospital gains financially if the number of days per admission is low, and the number of readmissions increases instead. However, this also increases the risk for low quality of the health care. For example, a low degree of readmissions of frail elderly patients shows that the hospital has successfully transitioned the patients to other health-care providers close to patients' homes. It is unclear what measure to use to estimate resource use: number of admissions, or average admission time. This will in turn affect how long time the patients are admitted at the hospital, and the number of admissions says very little about the need for, and use of, resources.

A lot of work is dedicated to decreasing the number of visits at the emergency department. The work involves a lot of collaboration with the ambulance department, the primary health care, the municipality health care, the special department for frail elderly people, and the department for guidance of people via telephone. All those collaborative activities are believed to affect the inflow of patients in a positive way, so that fewer patients seek emergency health care at the hospital.

Information from the registered data is used by politicians and decision makers at the hospital. Politicians use the data to get knowledge about the health care in order to allocate money to different areas and specialties. The health-care departments use the data to get knowledge about their performance, in order to develop and improve the health-care activities and processes. Especially, readmissions of patient are followed up, in order to get knowledge about to what extent the transitions to other health-care providers are successful. At the moment, the departments follow up their performance once a week, and the politicians intend to follow up once a month.

The top managers at the hospital scan the statistics a few times per year. Once per year, they decide how many patients each department could take care of, for each group of disease. In that way, they choose how to distribute the health-care beds and other resources among different patient groups. The total number of patients who are considered in the dimensioning is decided at the regional level, as a total budget for production of health care at each hospital. This dimensioning then affects whether patients need to be rejected or referred to another health-care provider, maybe to another health-care level, such as a primary health-care center.


**3. Effects of RL-TCLT on hippocampal structure and function**

The effects of RL-TCLT on the nervous system, neuronal repair, and improving cognition are growing, and have been well-documented in cellular, animal models, and human studies [8]. Since the past decade, the use of RL-TCLT as an advanced and non-invasive therapeutic method in several brain-related conditions has attracted interest from researchers in biomedical science, including those conditions or pathologies that manifest memory loss. Nevertheless, the underlying neural mechanisms are not well understood. The hippocampus is a brain structure of special importance in studying aging and cognitive decline, its main function is learning and memory [33]. It is a dorsoventrally elongated area, composed of the dentate gyrus (DG), the cornu ammonis (CA) fields CA1, CA2, and CA3, and the subiculum cortex [34]. The trisynaptic circuit is the main excitatory hippocampal synaptic pathway, formed by 3 neuronal groups: granule cells in the DG, and pyramidal neurons of the CA1 and CA3 [35]. This circuit receives inputs from the superficial layers of the entorhinal cortex via the perforant path to the DG. The DG projects to the CA3, which in turn projects to the CA1. Thus, CA1 projects to the deep layers of the entorhinal cortex, closing the circuit [33, 35, 36]. The hippocampus mediates recognition and spatial memory, by a highly regulated circuit with a high-energy demand [37]. Besides, is important to highlight that the hippocampus is highly susceptible to factors such as mitochondrial dysfunction, stress, inflammation, or physiological process such as aging, accumulating damage that gradually lead to a loss of hippocampal function [33].

Spatial memory gradually decreases with the age, since the hippocampus is critical for this type of memory, and the impairment of hippocampal neurons unequivocally results in spatial memory diminishing [37]. Studies from our and other groups have shown the reduced capacity of aged mice to learn and remember spatial tasks [4, 38, 39]. This is indicated by increased time to find a hidden platform in the Morris Water Maze (MWM) or a hidden chamber in the Barnes Maze (BM), two classic probes to evaluate hippocampus-dependent spatial memory [4, 22]. A report using the senescence-accelerated prone 8 (SAMP8) mice, a mouse model widely used to study oxidative impairment, and age-related brain damage, showed that RL-TCLT at 630 nm for two consecutive months prevents spatial memory loss in 5 month-old (mo) SAMP8 mice, and more importantly rescued the cognitive deficits in SAMP8 mice of 7 mo [40]. This last was accompanied by reduced ROS levels in the brain and increased activity of antioxidant enzymes such as catalase and formaldehyde dehydrogenase [40]. Similarly, TCLT with NIR laser at 810 nm applied in mice exposed to acute sleep deprivation showed reduced hippocampal oxidative damage, increasing the activity of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GPx) [16]. Additionally, several studies with cells, animals and in clinical trial conclude that RL-TCLT may have a potential effect on the brain since that has been observed that RL-TCTL protect nerve cells from a future impairment, reducing permanent neuronal damage and increasing their survival. For example, the treatment of K369I tau (K3) mice, a transgenic

mouse model of tauopathies and Alzheimer's Disease, with NIR (600–1000 nm) 20 times four weeks reveal a reduction in the size and number of amyloid-β plaques in the neocortex and hippocampus [41].

Besides, RL-TCLT may have a potential effect promoting both synaptogenesis and neurogenesis [42]. Both processes are essential to facilitating connectivity, neural regeneration, and generate structural changes that help to maintain existing neurons, and to encourage the growth of new neurons and synapses process [43, 44]. In this context, IR-light at 808 nm (350 mW/cm2 and 294 J) was applied in the scalp of a photothrombotic model of ischemic stroke in rats for seven days during 2-minute daily. The authors observed that IR-light therapy significantly attenuated behavioral deficits and infarct volume in cortical regions induced by photothrombotic stroke. This improvement was accompanied by neurogenesis and synaptogenesis, as is indicated by increased immunoreactivity of the proliferative and differentiation markers BrdU, Ki67, DCX, MAP2, spinophilin, and the synaptic marker synaptophysin [45]. Also, other clinical studies reveal positive effects of transcranial LED therapy on cerebral blood fluid (CBF) in patients in a vegetative state or with major depression and anxiety. LED treatment by 20 or 30 min per session, thrice per week over 6 weeks, or two times daily for over seventy days, with different wavelengths of 610, 627, and 810 nm increase CBF, improving cerebral vascular perfusion and reducing brain disorders [9, 46, 47]. Similarly, the application of TC-LLL therapy at 810-nm in mice model of cortical impact and traumatic brain injury reveal increased proliferating neural cells around the lesion, possibly activating regenerative mechanisms such as inducing neurogenesis in the dentate gyrus of the hippocampus [48]. Besides, they observed that the mice treated improved learning and memory reducing cognitive impairment [48, 49].

Thus, while its positive effects have been demonstrated countless times in animal models, they have yet to be proven in broad-scope clinical testing. However, the research that does exist is very promising, strongly indicating that RL-TCLT could be a viable treatment for a broad range of neurological diseases including stroke, traumatic brain injury, Parkinson's disease, Alzheimer's disease, and depression, in addition to providing cognitive enhancement for healthy subjects of advanced age that manifest cognitive impairment.


**4. Conclusions**

We could highlight some direct results in a summary of the two analyses carried out in this article on texts and images.

Based on the doctrinal tradition established by the Fathers and theologians in this regard, numerous medieval hymnographers composed countless canticles, antiphons, and liturgical hymns in which they exalt Mary as the open "gate of heaven" (*ianua Coeli*), "door of Paradise" (*porta Paradisi*), and as its equivalent concepts "mediator", "intercessor", or other similar titles referring to Mary's powerful mediation before her divine Son in favor of humankind.

Many hymns underline that Mary's saving faculty derives from her privilege as the virginal mother of God the Son. So, being the mother of the Savior, who is also the Supreme Judge, she is in the best condition to intercede on behalf of the human gender.

On the other hand, those ideas of the liturgical hymns—and, of course, those of the Church Fathers and the theologians who inspired them—were reflected during the Middle Ages and the Renaissance in a series of sculptural and pictorial artworks that show the Virgin Mary as the gate of Heaven, and as a mediator before God in favor of the faithful.

In this article, we bring to light three iconic modalities in which Mary is represented as a mediator and intercessor of humanity under the metaphor of "gate of Heaven or door of Paradise": the *Deësis* or *Deisis* in the scene of the Last Judgment, the figure of the Virgin with her Child at the entrance of the temple, and third, the Virgin framed by a door or arch in the scenes of the Annunciation.

Medieval Christianity nurtured the faith of those mostly illiterate populations with two great resources: words and images. The words were expressed, above all, in the sermons of the ecclesiastics in the temple, and these, in turn, fed on the texts of the Bible (Old and New Testament), the Church Fathers, and theologians. To these primary textual expressions of the priests before the illiterate faithful were later added the lyrics of the songs, antiphons, and liturgical hymns that, although written in Latin and also being indecipherable for the illiterate majority, could be explained by the ecclesiastics on the occasion of the celebration of the Mass, or during processions, ceremonies, and devotional acts. Based on such explanations, it seems reasonable to conjecture that few of those uneducated medieval Christians would be ignorant of the basic meanings of such popular antiphons as the *Salve Regina* or the *Regina Coeli laetare*, even if they were not able to read/translate each of their Latin words.

One last important conclusion is necessary: these statements of the liturgical hymns and those sculptural and pictorial images centered around the metaphor of Mary as *porta Paradisi* are in perfect harmony. After all, what the liturgical hymns poetically proclaimed reflected the thousand-year-old exegetical tradition of the Church Fathers and medieval theologians on Mary's universal mediation symbolized by the *ianua Coeli* or *porta Paradisi* metaphors. Additionally, on the other hand, the ecclesiastical hierarchy could not miss the opportunity to "catechize" its illiterate faithful through that "catechism in stone or paint" materialized in those sculptures and paintings that iconographically represented Mary as the gate of Heaven.

Thus, both texts and images affect the spirit of the believer with the same catechetical effectiveness. In this way, after seeing the material sculptures or paintings of the Virgin "gate of Heaven" upon entering the temple and hearing the immaterial enouncements of the liturgical hymns sung there, the faithful could be super-assured in a solid thesis of the Christian faith: Mary is the effective mediator before God, and the believer must resort to her to obtain goods on this Earth and eternal salvation in Heaven.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The author declares no conflict of interest.


*6.3. Simulation*

A simulation of the full system, formed by a 2LVSI operating as central inverter grid-tied PV system, and ESS formed by a Battery ESS (BESS) and an Isolated Bidirectional Boost Converter [35], is shown in Figure 9. The control strategies previously described in Section 6.2 were applied. It must be noted that the ESS was undersized to allow its control system to be tested in all possible scenarios and display those results in a single figure; this was performed considering the dynamics of the systems and an ESS operating range of 20–80% of the SoC. Several irradiance conditions were tested in the simulations, all with PV cell temperature of 298.15 ◦K.

The PV plant parameters correspond to those presented in Table 1, while Table 4 presents the parameters of the converters and ESS. To calculate per-unit values in Figure 9, the following base values were considered: *G*base = 1000 W/m2, *P*base = 1540 kW, *i*base = 3.767 kA and *v*base = 800 V.

**Table 4.** Simulation parameters.

**Figure 9.** PV plant with ESS connected at the DC-link simulation results: (**a**) irradiance in kW/m2; (**b**) DC-link voltage and reference; (**c**) generated PV power (*P*pv), available PV power without considering clipping limitation (*P*mpp (available PV power)) and inverter power (*P*inv (inverter)); (**d**) ESS power (*P*ess); (**e**) ESS SoC (*SoC*); and (**f**) grid currents (*i*u, *i*v and *i*w) and phase u voltage (*v*u).

From 0 to 0.03 s (Case I), the system is operating at a power rating below *P*clip (1540 kW) and the ESS is at a SoC level of 20%, hence ESS power reference is zero. From 0.03 to 0.14 s (Case II), a step in solar irradiance causes the PV power (*P*pv) to reach *P*clip, since *SoC* < 80%, *P*∗ess power reference is set to *P*∗ess = *P*pre − *P*mpp (and *i*∗ess = *P*∗ess/*v*pv), which reduces power flowing through the inverter (*P*inv) enabling MPP tracking. From 0.14 to 0.20 s (Case III), a step down in solar irradiance causes the PV power (*P*pv) to be lower than *P*clip, but, since *P*pre < *P*pv < *P*clip, the ESS power reference (*P*∗ess) is set to zero. From 0.2 to 0.25 s (Case II), a second step up in irradiance generates for the PV power to surpass *P*clip, the system behaves exactly as from 0.03 to 0.14 s. Once the ESS reaches 80% of the SoC, the ESS stops drawing power and the inverter losses momentarily the capability to track the MPP (Case IV from 0.25 to 0.28 s). Note that the ESS was undersized, to show the behavior of the system

when reaching its maximum and minimum permitted SoC. From 0.28 to 42 s (Case V), a step down in solar irradiance causes the PV power to be below pre clipping level (*P*pv < *P*pre < *P*clip), and the ESS releases power towards the DC-link (*P*∗ess = *P*pre − *P*mpp) and through the inverter into the grid. From 0.42 to 0.5 s, the ESS reaches 20% of the SoC, hence power flow from ESS towards the DC-link is stopped and the system goes back to operating in Case I; in this part, grid currents show high harmonic content due the comparison of power flowing to the grid and power required to generate voltage steps to perform MPPT. In a real case, the MPPT period is longer, hence harmonic content would be lower.

Note that the instantaneous power balance is given by Equation (8), where the corresponding terms are given by Equations (9)–(11). This can also be verified in power curves shown in Figure 9c,d. Variables *v*gd, *v*gq, *i*d and *i*q in Equation (9) correspond to the grid voltage and current in rotational coordinates.

$$P\_{\rm inv}(t) \quad = \quad P\_{\rm pv}(t) + P\_{\rm ess}(t) \tag{8}$$

$$P\_{\rm inv}(t) \quad = \ \frac{3}{2} \cdot \left( v\_{\rm gd}(t) \cdot i\_{\rm d}(t) + v\_{\rm gq}(t) \cdot i\_{\rm q(t)} \right) \tag{9}$$

$$P\_{\rm PV}(t) \quad = \ i\_{\rm PV}(t) \cdot v\_{\rm PV}(t) \tag{10}$$

$$P\_{\rm ess}(t) \quad = \ i\_{\rm ess}(t) \cdot \upsilon\_{\rm pv}(t) \tag{11}$$