**4. Possible mechanism of action (MoA) of BMMSCs for treatment of osteoarthritis**

The pathophysiology of OA is characterized by degradation of hyaline cartilage causing narrowing of joint space leading to subchondral sclerosis, subchondral cysts, hypertrophic chondrocytes, and formation of osteophytes. The friction caused by the rubbing of joints results in chronic pain in OA patients [24]. Degeneration of cartilage extracellular matrix (ECM) may be caused due to the increase in the levels of proteolytic enzymes such as matrix metalloproteases (MMPs) and aggrecanases mediated by IL-1β and TNFα [25]. BMMSCs express a wide range of properties that are anticipated to be beneficial for treating genetic, mechanical, and age-related degeneration in diseases such as OA. In our previous publication, we have in detail attempted to deduce the possible mechanism of action (MoA) of allogeneic pooled BMMSC population [25]. Briefly, BMMSCs are known to be immunomodulatory in nature, primarily because of their potential to significantly suppress the proliferation of inflammatory T cells, monocytes, and dendritic cells either by direct cell-to-cell contact. In addition, they secrete a wide range of anti-inflammatory molecules such as PGE-2, IDO, IL1Ra, and IL-10 [26, 27]. BMMSCs influence the local osteoarthritic microenvironment by stimulating

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

**2. Current treatment options for osteoarthritis**

chondrocytes and synoviocytes [5]. Further, increase in the levels of interferon γ (IFNγ) in the joint worsens the inflammatory state and structure of the joint leading to degradation of proteoglycans such as sulfated glycosaminoglycans (sGAG) [5, 6].

Currently, pain in OA is pharmacologically managed using nonsteroidal antiinflammatory drugs (NSAIDS), opioids, and analgesics. Corticosteroid injections have also been used for relieving severe pain in OA patients. Recent attempts have been made to use TNFα blockers as recent studies have proven the significant role of TNFα in contribution to the pathogenesis of OA [7]. Research by several groups has implicated the role of nerve growth factor (NGF) and its binding to tropomyosin receptor kinase A (trk A) which leads to downstream signaling and activation of peripheral and central pain molecules causing severe pain. The therapeutic efficiency of anti-NGF antibodies to block NGF or its antagonists has been studied by several groups for relieving pain. The pain-relieving effects of anti-NGF antibodies fasinumab and fulranumab manufactured by Regeneron Pharmaceuticals and Janssen Pharmaceutica, respectively, have been evaluated in phase III clinical trials [8]. In addition to pain relief, efforts have been made to halt further cartilage damage using slow-acting symptomatic drugs such as chondroitin sulfate and glucosamine sulfate. Orally administered chondroitin and glucosamine have shown to relieve joint pain equivalently compared to NSAIDs. These molecules, intact or broken, could be absorbed into the matrix of the joint and prevent cartilage degeneration. Although glucosamine and chondroitin sulfate have been clinically proven to be safe, their therapeutic efficacy in protecting the cartilage matrix was found to the variable [9]. In grade 4 OA (Kellgren and Lawrence classifications), patients are advised to opt for total knee replacement surgery [10]. Alternatively, autologous chondrocyte implantation (ACI) has been suggested and reported to be successful. In the ACI method, the chondrocytes from patients are taken, culture-expanded in vitro, and then implanted back into the knees of patients. This procedure is invasive and has a lesser success rate than total knee replacement surgeries [11]. Apart from ACI, the efficacy of autologous platelet-rich plasma (PRP) in providing pain relief and promoting cartilage regeneration has been recently investigated by several groups [12]. The PRP is rich in platelets that secrete several growth factors and cytokines such as platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE-2) [13]. Several research groups have reported that intra-articular injections of PRP primarily reduced inflammation mediated by PGE-2, HGF, and IGF-1. IGF-1 synthesized and secreted by platelets is shown to prevent leukocyte infiltration into the joint space, thereby reducing the levels of IL-1β and TNFα in the synovial fluid [13]. Overall intra-articular injection of PRP has been shown to maintain joint homeostasis. However, clinical trial data suggest that the effect of PRP seems to last for only 3 weeks and thereafter reduces. The symptoms of OA were seen to relapse after a period of 1 year. Although promising results were observed using PRP in the hydrogel, chitosan, or hyaluronic acid (HA) scaffolds [14],

efficacy is yet to be shown in elaborate randomized clinical trials (RCTs).

The history of mesenchymal stromal/stem cells (MSCs) dates back to 1960 when seminal studies conducted by Friedenstein showed the isolation of MSCs from bone

**52**

**3. Mesenchymal stromal cells**

resident chondrogenic progenitor cells and promote their differentiation into mature chondrocytes mediated by secretion of bone morphogenetic proteins (BMPs) and TGFβ1 [28]. BMMSCs are known to differentiate into chondrocytes in vitro using differentiation cues such as BMP-7 and TGFβ1. A similar mechanism could be involved in the differentiation of BMMSCs in vivo. With the increase in the levels of BMP-7 and TGFβ1 in the local joint milieu, mediated by a change in expression of master regulatory genes such as Sox9, HoxA, HoxD, and Gli3, BMMSCs could differentiate into chondrogenic progenitor cells (CPCs) in vivo. The CPCs further differentiate into chondroblasts characterized by definitive upregulation of collagen types II B, IX, and XI. Subsequently, the CPCs differentiate into mature chondrocytes regulated by balanced expression of collagen X (Col X) and synthesize the secretion of collagen II which is made of sGAG building blocks which maintain the structural integrity of hyaline cartilage [25]. Very high expression of collagen X has been linked to hypertrophy of chondrocytes and formation of fibrous cartilage, and thus a regulated expression of Col X would likely result in deposition of hyaline cartilage [29]. From the above-described multimodal MoA, it is clear that BMMSCs are an ideal cell population which could contribute significantly for an effective treatment of OA.

## **5. Advantages of using a pooled human BMMSC (phBMMSC, Stempeucel®) product for treating osteoarthritis**

In the current therapeutic scenario, the common practice is to screen several individual donors, isolate MSCs, and characterize them based on their key characteristics such as their surface marker expression, tri-lineage differentiation potential, and immunomodulatory and paracrine properties [30–32]. It is inevitable that a product that is manufactured using a master cell bank (MCB) made from a single donor will result in exhaustion. Successively, a product that is made using another single donor MSC bank, although presumably similar in basic characteristics qualifying the identity and safety criteria, may not have the same functional attributes which may lead to varied therapeutic outcomes. Eminent scientific groups have demonstrated donor-todonor variability in properties of MSCs such as their clonogenicity, growth kinetics, and differentiation potential [33]. A comparative analysis of five different BMMSC populations showed significant variation in the proteomic profile of these cells. Only 13% similarity in the proteomic profile which included transcriptional and translational regulators, kinases, receptor proteins, and cytokines between the five BMMSC populations was found. A maximum of 72% similarity in the proteome was observed between two of the five analyzed cell populations [34]. Disparities in clinical trial outcomes have been reported where BMMSCs derived from single donors have been used. A steroid-refractory acute graft-versus-host disease (SR-aGvHD) clinical trial conducted in both children (n = 25) and adults (n = 30) using BMMSC products derived from 92 HLA-matched and HLA-mismatched donors resulted in only 50% overall durable complete response, while the remaining patients did not respond or partially responded to the treatment [35]. Similar variations with limited response rates were observed in a phase III GvHD trial conducted by Osiris Therapeutics using Prochymal® with only 35% complete response rate compared to 30% in the placebo arm [36]. It has been suggested that improper selection of a BM donor and making a single donor-derived cell product could lead to substantial variations in therapeutic outcomes [37]. In order to challenge this issue, some scientific groups have suggested pooling of BMMSCs from two or more donors in order to compensate for the variation and balance the properties between different donor cell populations. Samuelsson et al. showed that a two- or three-donor pooled BMMSC product could

**55**

**Figure 1.**

**osteoarthritis**

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

optimize the immunosuppressive properties of these cells in vitro [38]. Later, Kuçi et al. showed substantial variability in the immunosuppressive properties of individual donor-derived BMMSCs (n = 8). On the contrary, a mesenchymal end product (MEP) made by pooling BMMNCs from eight donors resulted in a cell population that consistently suppressed an MLR in vitro [39]. Subsequently, they went on to conduct a multicentric SR-aGvHD clinical trial in 51 children and 18 adults using MEP/MSC Frankfurt am Main (MSC-FFM, Obnitix®) cells and observed 83% overall response (complete response, 32%; partial response, 51%) [40]. At Stempeutics Research Pvt. Ltd., we were the first group to develop an allogeneic pooled human BMMSC product called Stempeucel® using an established, robust pooling protocol and a two-tier manufacturing and banking system as previously described [41, 42]. Recently, we have published our comprehensive studies including in vitro chondrogenic properties and preclinical and clinical findings establishing the efficacy and safety of using Stempeucel® for the treatment of OA of the knee joint [43]. In this study, we found that several manufactured batches of Stempeucel®, when differentiated into the chondrocyte lineage, downregulated the expression of the gene Sox9 and upregulated the expression of collagen type 2A (Col2A) gene confirming their differentiation into the chondrogenic lineage. The same Stempeucel® batches synthesized substantial levels of sGAG (30 ± 1.8 μg/μg GAG/DNA) which were estimated using a dimethylmethylene blue-based biochemical assay kit (**Figure 1**). These properties indicate that

*Chondrogenic potency assessment using quantification of sGAG in 16 batches of Stempeucel® cryopreserved in PlasmaLyte A-based cryopreservation solution and 4 batches of Stempeucel® cryopreserved in CS 5.*

Stempeucel® could be a potential treatment option for treating OA.

**6. Development of a potency assay for Stempeucel® intended to treat** 

The US Food and Drug Administration (USFDA) describes potency assays as "The specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

**Figure 1.**

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

cantly for an effective treatment of OA.

resident chondrogenic progenitor cells and promote their differentiation into mature chondrocytes mediated by secretion of bone morphogenetic proteins (BMPs) and TGFβ1 [28]. BMMSCs are known to differentiate into chondrocytes in vitro using differentiation cues such as BMP-7 and TGFβ1. A similar mechanism could be involved in the differentiation of BMMSCs in vivo. With the increase in the levels of BMP-7 and TGFβ1 in the local joint milieu, mediated by a change in expression of master regulatory genes such as Sox9, HoxA, HoxD, and Gli3, BMMSCs could differentiate into chondrogenic progenitor cells (CPCs) in vivo. The CPCs further differentiate into chondroblasts characterized by definitive upregulation of collagen types II B, IX, and XI. Subsequently, the CPCs differentiate into mature chondrocytes regulated by balanced expression of collagen X (Col X) and synthesize the secretion of collagen II which is made of sGAG building blocks which maintain the structural integrity of hyaline cartilage [25]. Very high expression of collagen X has been linked to hypertrophy of chondrocytes and formation of fibrous cartilage, and thus a regulated expression of Col X would likely result in deposition of hyaline cartilage [29]. From the above-described multimodal MoA, it is clear that BMMSCs are an ideal cell population which could contribute signifi-

**5. Advantages of using a pooled human BMMSC (phBMMSC,** 

In the current therapeutic scenario, the common practice is to screen several individual donors, isolate MSCs, and characterize them based on their key characteristics such as their surface marker expression, tri-lineage differentiation potential, and immunomodulatory and paracrine properties [30–32]. It is inevitable that a product that is manufactured using a master cell bank (MCB) made from a single donor will result in exhaustion. Successively, a product that is made using another single donor MSC bank, although presumably similar in basic characteristics qualifying the identity and safety criteria, may not have the same functional attributes which may lead to varied therapeutic outcomes. Eminent scientific groups have demonstrated donor-todonor variability in properties of MSCs such as their clonogenicity, growth kinetics, and differentiation potential [33]. A comparative analysis of five different BMMSC populations showed significant variation in the proteomic profile of these cells. Only 13% similarity in the proteomic profile which included transcriptional and translational regulators, kinases, receptor proteins, and cytokines between the five BMMSC populations was found. A maximum of 72% similarity in the proteome was observed between two of the five analyzed cell populations [34]. Disparities in clinical trial outcomes have been reported where BMMSCs derived from single donors have been used. A steroid-refractory acute graft-versus-host disease (SR-aGvHD) clinical trial conducted in both children (n = 25) and adults (n = 30) using BMMSC products derived from 92 HLA-matched and HLA-mismatched donors resulted in only 50% overall durable complete response, while the remaining patients did not respond or partially responded to the treatment [35]. Similar variations with limited response rates were observed in a phase III GvHD trial conducted by Osiris Therapeutics using Prochymal® with only 35% complete response rate compared to 30% in the placebo arm [36]. It has been suggested that improper selection of a BM donor and making a single donor-derived cell product could lead to substantial variations in therapeutic outcomes [37]. In order to challenge this issue, some scientific groups have suggested pooling of BMMSCs from two or more donors in order to compensate for the variation and balance the properties between different donor cell populations. Samuelsson et al. showed that a two- or three-donor pooled BMMSC product could

**Stempeucel®) product for treating osteoarthritis**

**54**

*Chondrogenic potency assessment using quantification of sGAG in 16 batches of Stempeucel® cryopreserved in PlasmaLyte A-based cryopreservation solution and 4 batches of Stempeucel® cryopreserved in CS 5.*

optimize the immunosuppressive properties of these cells in vitro [38]. Later, Kuçi et al. showed substantial variability in the immunosuppressive properties of individual donor-derived BMMSCs (n = 8). On the contrary, a mesenchymal end product (MEP) made by pooling BMMNCs from eight donors resulted in a cell population that consistently suppressed an MLR in vitro [39]. Subsequently, they went on to conduct a multicentric SR-aGvHD clinical trial in 51 children and 18 adults using MEP/MSC Frankfurt am Main (MSC-FFM, Obnitix®) cells and observed 83% overall response (complete response, 32%; partial response, 51%) [40]. At Stempeutics Research Pvt. Ltd., we were the first group to develop an allogeneic pooled human BMMSC product called Stempeucel® using an established, robust pooling protocol and a two-tier manufacturing and banking system as previously described [41, 42]. Recently, we have published our comprehensive studies including in vitro chondrogenic properties and preclinical and clinical findings establishing the efficacy and safety of using Stempeucel® for the treatment of OA of the knee joint [43]. In this study, we found that several manufactured batches of Stempeucel®, when differentiated into the chondrocyte lineage, downregulated the expression of the gene Sox9 and upregulated the expression of collagen type 2A (Col2A) gene confirming their differentiation into the chondrogenic lineage. The same Stempeucel® batches synthesized substantial levels of sGAG (30 ± 1.8 μg/μg GAG/DNA) which were estimated using a dimethylmethylene blue-based biochemical assay kit (**Figure 1**). These properties indicate that Stempeucel® could be a potential treatment option for treating OA.

## **6. Development of a potency assay for Stempeucel® intended to treat osteoarthritis**

The US Food and Drug Administration (USFDA) describes potency assays as "The specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the

product in the manner intended, to effect a given result" (US-FDA, 21 CFR 600). For any cell therapy product (CTP) intended to be used for a particular indication, a specific, quantifiable, potency test or array must be developed. The development of a potency assay must begin with in vitro and preclinical studies based on the MoA of the CTP. The confirmation of the assay or the identified marker must be evaluated in every large-scale manufactured batch of the CTP during the progress of the phase I and phase II clinical studies. A quantifiable range for the potency test must be defined and implemented during the course of phase III clinical trial [44]. In order to predict the efficacy of a CTP, either in vitro biochemical assays or biological assays or in vivo biological assessment could be implemented. For example, a company called TiGenix (Leuven, Belgium) has developed and adopted an assay matrix where an ex vivo polymerase chain reaction (PCR) array for autologous chondrocytes (ChondroCelect) is performed and ectopic cartilage formation is correlated to the histology sections of an orthotopic goat model where ChondroCelect is implanted [45, 46]. Jeong et al. have demonstrated that thrombospondin-2 (TSP-2) could be an effective marker to predict the chondrogenic efficiency of umbilical cord-derived MSCs (UC-MSCs). They demonstrated that UC-MSCs, through the TSP-2 secretion, can promote chondrogenesis via PKCa, ERK, P38/MAPK, and Notch signaling pathways [47]. Recently, another group estimated the levels of TSP-2 to evaluate the chondrogenic potency of a UC-MSC product (Cellistem®OA, Cells for Cells, Brazil) intended to be used in phase I/phase II RCT for knee OA [48]. Other scientific groups have shown that autologous culture-expanded chondrocytes could be embedded in collagen-1 and injected subcutaneously in nude mice to predict the potency of several bioactive molecules in promoting chondrogenesis [49]. For the first time, we have developed a chondrogenic potency assay for an allogeneic pooled bone marrow-derived MSC product (phBMMSCs, Stempeucel®). Preliminarily, we culture-expanded and differentiated several Stempeucel® batches into the chondrogenic lineage using commercially available differentiation assay kits (Thermo Fisher Scientific, USA). To confirm the differentiation, we evaluated the Col2A mRNA expression in differentiated cells and compared them with the undifferentiated control cells. After observing a significant increase in the Col2A expression of differentiated cells, we enzymatically digested both the differentiated and undifferentiated cells to quantify the levels of sGAG synthesized by these cells using a 1,9-dimethylmethylene blue (DMMB)-based assay kit (Blyscan, Biocolor, UK). We further normalized the levels of sGAG with the amount of DNA from the same number of cells. We evaluated the sGAG levels in 20 batches of Stempeucel® of which 16 batches were cryopreserved in our older formulation (10% dimethyl sulfoxide (DMSO), 5% human serum albumin (HSA) and PlasmaLyte A) and also four batches of Stempeucel® cryopreserved in a new cGMP grade CryoStor 5 solution (CS5, BioLife Solutions). We observed a significant and consistent increase in the levels of sGAG in the differentiated cells compared to the undifferentiated cells (undifferentiated, 11.9 ± 4.6 GAG/DNA (μg/μg); differentiated, 31 ± 8.6 GAG/DNA (μg/μg; P < 0.0001; n = 20)) (**Figure 1**). Based on our results, we propose that the sGAG assay is a simple, quantifiable, and robust potency assay which could also be a part of a bigger potency assay matrix to predict the chondrogenic potency of therapeutic cells intended to treat cartilage defects.

## **7. Preclinical efficacy studies in OA**

Many studies have demonstrated that MSCs are nontoxic and non-tumorigenic when tested in various animal models [50, 51]. Prior to evaluating the efficacy of Stempeucel® in an appropriate preclinical model of OA, we had earlier evaluated the preclinical safety and toxicity of Stempeucel® in rodent and non-rodent

**57**

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

**dose**

10 × 106

10.5 × 106 Autologous (horse) BMMSC

10 × 106 Autologous (sheep) BMMSC

20 × 106 Autologous (sheep) BMMSC

1 × 105

1 × 106

0.5 × 106 Autologous (rat) BMMSC

or 1.3 × 106 Xenogeneic (human) pooled BMMSC

Autologous (goat) BMMSC + HA

 Xenogeneic (human) BMMSC

 Autologous (rabbit) BMMSC

 Allogeneic (rabbit) BMMSC

 Allogeneic (mice) BMMSC

 Allogeneic (rat) BMMSC

Allogeneic (rat) BMMSC

**Vehicle Study** 

HA 12 and

HA/PBS 1, 3, and

PBS 0, 1, 4,

HA 6 and

HA 3 and

Culture medium

Saline/mouse albumin

Culture medium **duration time points**

26 weeks

5 weeks

8, and 12 weeks

4 and 6 weeks

12 weeks

9 weeks

3 and 6 weeks

12 weeks

PBS 3 weeks [54]

PlasmaLyte A 4, 8, and

8 weeks [52]

Saline 10 weeks [61]

PBS 8 weeks [63]

**Reference**

[65]

[66]

[64]

[55]

[56]

[67]

[53]

[43]

**Animal OA model Cell type and** 

meniscectomy

Pig Spontaneous 7 × 106

meniscectomy

meniscectomy

Rabbit ACLT 1 × 106

Rabbit ACLT 1 × 106

Rat MIA injection 3.5 × 106

meniscectomy

meniscectomy

Rat MIA injection 0.6 × 106

models. In the same study, we evaluated the feasibility of multiple routes of cell injection. Tumorigenic analysis in severe combined immunodeficient (SCID) mice showed that Stempeucel® is non-tumorigenic. In addition, the biodistribution kinetics of CM-DiI labeled Stempeucel® in the systemic circulation and also in

It is important to demonstrate the efficacy of any cell therapy product in an animal model of disease before administrating the product in humans with the same disease. It is imperative to determine the suitability of using animal stem cells in animals or human stem cells in immunocompromised/immunocompetent animals. A common regulatory requirement is to have animal data for the same test product that is intended to be tested in humans. In our recently published work, we evaluated the efficacy of Stempeucel® in a monosodium iodoacetate (MIA)-induced

muscle tissue were studied in both rats and mice [51].

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

Goat ACLT-

Sheep ACLT-

Sheep Bilateral

Mouse Closed tibial plateau fracture

Rat ACLT-

Rat ACLT-

*OA preclinical studies using BMMSCs.*

Horse Arthroscopic surgery

**Author/ year**

Murphy et al. (2003)

Frisbie et al. (2009)

Sato et al. (2012)

Song et al. (2014)

Delling et al. (2015)

Singh et al. (2014)

Chiang et al. (2016)

Diekman et al. (2013)

Suhaeb et al. (2012)

Kim et al. (2014)

Yang et al. (2015)

Gupta et al. (2016)

**Table 1.**


*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

#### **Table 1.**

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

product in the manner intended, to effect a given result" (US-FDA, 21 CFR 600). For any cell therapy product (CTP) intended to be used for a particular indication, a specific, quantifiable, potency test or array must be developed. The development of a potency assay must begin with in vitro and preclinical studies based on the MoA of the CTP. The confirmation of the assay or the identified marker must be evaluated in every large-scale manufactured batch of the CTP during the progress of the phase I and phase II clinical studies. A quantifiable range for the potency test must be defined and implemented during the course of phase III clinical trial [44]. In order to predict the efficacy of a CTP, either in vitro biochemical assays or biological assays or in vivo biological assessment could be implemented. For example, a company called TiGenix (Leuven, Belgium) has developed and adopted an assay matrix where an ex vivo polymerase chain reaction (PCR) array for autologous chondrocytes (ChondroCelect) is performed and ectopic cartilage formation is correlated to the histology sections of an orthotopic goat model where ChondroCelect is implanted [45, 46]. Jeong et al. have demonstrated that thrombospondin-2 (TSP-2) could be an effective marker to predict the chondrogenic efficiency of umbilical cord-derived MSCs (UC-MSCs). They demonstrated that UC-MSCs, through the TSP-2 secretion, can promote chondrogenesis via PKCa, ERK, P38/MAPK, and Notch signaling pathways [47]. Recently, another group estimated the levels of TSP-2 to evaluate the chondrogenic potency of a UC-MSC product (Cellistem®OA, Cells for Cells, Brazil) intended to be used in phase I/phase II RCT for knee OA [48]. Other scientific groups have shown that autologous culture-expanded chondrocytes could be embedded in collagen-1 and injected subcutaneously in nude mice to predict the potency of several bioactive molecules in promoting chondrogenesis [49]. For the first time, we have developed a chondrogenic potency assay for an allogeneic pooled bone marrow-derived MSC product (phBMMSCs, Stempeucel®). Preliminarily, we culture-expanded and differentiated several Stempeucel® batches into the chondrogenic lineage using commercially available differentiation assay kits (Thermo Fisher Scientific, USA). To confirm the differentiation, we evaluated the Col2A mRNA expression in differentiated cells and compared them with the undifferentiated control cells. After observing a significant increase in the Col2A expression of differentiated cells, we enzymatically digested both the differentiated and undifferentiated cells to quantify the levels of sGAG synthesized by these cells using a 1,9-dimethylmethylene blue (DMMB)-based assay kit (Blyscan, Biocolor, UK). We further normalized the levels of sGAG with the amount of DNA from the same number of cells. We evaluated the sGAG levels in 20 batches of Stempeucel® of which 16 batches were cryopreserved in our older formulation (10% dimethyl sulfoxide (DMSO), 5% human serum albumin (HSA) and PlasmaLyte A) and also four batches of Stempeucel® cryopreserved in a new cGMP grade CryoStor 5 solution (CS5, BioLife Solutions). We observed a significant and consistent increase in the levels of sGAG in the differentiated cells compared to the undifferentiated cells (undifferentiated, 11.9 ± 4.6 GAG/DNA (μg/μg); differentiated, 31 ± 8.6 GAG/DNA (μg/μg; P < 0.0001; n = 20)) (**Figure 1**). Based on our results, we propose that the sGAG assay is a simple, quantifiable, and robust potency assay which could also be a part of a bigger potency assay matrix to predict the chondrogenic potency of thera-

**56**

peutic cells intended to treat cartilage defects.

Many studies have demonstrated that MSCs are nontoxic and non-tumorigenic when tested in various animal models [50, 51]. Prior to evaluating the efficacy of Stempeucel® in an appropriate preclinical model of OA, we had earlier evaluated the preclinical safety and toxicity of Stempeucel® in rodent and non-rodent

**7. Preclinical efficacy studies in OA**

*OA preclinical studies using BMMSCs.*

models. In the same study, we evaluated the feasibility of multiple routes of cell injection. Tumorigenic analysis in severe combined immunodeficient (SCID) mice showed that Stempeucel® is non-tumorigenic. In addition, the biodistribution kinetics of CM-DiI labeled Stempeucel® in the systemic circulation and also in muscle tissue were studied in both rats and mice [51].

It is important to demonstrate the efficacy of any cell therapy product in an animal model of disease before administrating the product in humans with the same disease. It is imperative to determine the suitability of using animal stem cells in animals or human stem cells in immunocompromised/immunocompetent animals. A common regulatory requirement is to have animal data for the same test product that is intended to be tested in humans. In our recently published work, we evaluated the efficacy of Stempeucel® in a monosodium iodoacetate (MIA)-induced

OA model in Wistar rats. We demonstrated the dose-dependent efficacy of two Stempeucel® doses of 0.65 × 106 (25 × 106 human equivalent dose, HED) and 1.3 × 106 (50 × 106 HED) followed by an injection of hyaluronic acid (HA). A significant dose-dependent reduction in pain scores was observed in both low and high Stempeucel® doses compared to the HA alone and disease control group. Histological evaluation of joint tissue sections in all study groups showed significant improvement in proteoglycan staining in both low and high Stempeucel® administered groups indicating significant regeneration of the cartilage in both groups compared to the HA alone and disease control groups [43].

Similar to the animal model we used, other scientific groups have created articular cartilage defects in small animals, such as mice [52], rats [43, 53, 54], and rabbits [55, 56]. Smaller animal models are cost-effective and easy to house, and rodents are available in a variety of genetically modified strains with minimal biological variability [57, 58]. However, the small joint size, thin cartilage, altered biomechanics, and increased spontaneous intrinsic healing hamper the study of the regenerative capacity of stem cells and these mechanisms of healing which cannot be fully extrapolated to human cartilage repair [59, 60]. Rodents have mainly been used to assess the chondrogenesis of cell-based therapies by subcutaneous, intramuscular, and intra-articular implantations of cells [60]. Of all small animals, the rabbit model is the most utilized model in cartilage regeneration studies because of the slightly larger knee joint size than rodents [55, 56]. Despite their limited translational capacity, small animals can be very useful as a proof-of-principle study and to assess therapy safety before moving on to preclinical studies using larger animals [60].

Large animal models play a more substantial role in translational research because of a larger joint size and thicker cartilage; however, their preclinical use is often hindered by high costs and difficulties in animal handling. A variety of large animal models have been used to investigate cartilage repair strategies, including horses [61], dogs [62], sheep [63, 64], goats [65], and pigs [66], each with their own strengths and limitations. We have listed some relevant published studies which have used autologous, allogeneic, or xenogeneic BMMSCs to treat OA induced by various methods (**Table 1**).

Based on the positive efficacy outcomes of our preclinical study, subsequently, we demonstrated the safety and optimal dose for efficacy in a phBMMSC product, Stempeucel®, in a randomized, double-blind, placebo-controlled dose-finding phase II clinical trial in Indian patients [43].

### **8. Clinical trials in osteoarthritis of the knee joint**

#### **8.1 Safety of mesenchymal stromal cells in clinical trials**

Lalu MM et al. conducted a systematic review of clinical trials that examined the use of MSCs to evaluate their safety [68]. A total of 36 studies having 1012 participants with different clinical conditions was evaluated. Eight studies were randomized control trials (RCTs) and enrolled 321 participants. Only prospective clinical trials that used the intravenous or intra-arterial route of administration in different age groups were analyzed. Meta-analysis did not detect an association with MSC administration and acute infusional toxicity, organ system complications, infection, and death. There was a significant association between MSCs and transient fever at or shortly after MSC administration which was not associated with long-term sequelae. Most importantly, the meta-analysis showed no serious adverse event due to the administration of MSCs and specifically found no association between MSCs and tumor formation. In another study, Peeters et al. [69] did a systemic review of

**59**

this chapter:

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

the safety of intra-articular administration of culture-expanded stem cells. A total of 844 procedures (mean follow-up of 21 months) was analyzed. Four SAEs were reported—one infection following bone marrow aspiration (BMA) that resolved with antibiotics, one pulmonary embolism after 2 weeks of BMA, and two adverse events not related to the therapy. Other adverse events documented were increased pain/swelling and dehydration after BMA. In another review, a recent analysis of adverse events (AEs) in 2372 orthopedic patients treated with autologous stem cell therapies and followed up for 2.2 years has been published [70]. The common AEs reported included post-procedure pain and pain due to progressive degenerative joint disease in under 4% of the population. Hence, we can conclude that the systemic administration of MSC including intra-articular administration is safe.

**8.2 Efficacy of stem cells including mesenchymal stromal cells in clinical trials** 

Several clinical trials have been conducted using bone marrow mononuclear cells, adipose tissue-derived stromal vascular fraction (AD-SVF), adipose tissuederived mesenchymal stromal cells (AD-MSCs), or bone marrow-derived mesenchymal stromal cells (BMMSCs) in OA of the knee joint. The list of the published clinical trials in chronological order is given in **Table 2**. Administration of the cells has been fairly standardized, with the cells being administered either directly intraarticularly or under ultrasound guidance. Few trials have been conducted using the arthroscopic method of administration with direct implantation of the cells alone or

The first clinical study has been published way back in 2002 by Wakitani et al. [71]. In this study of 12 patients who underwent high tibial osteotomy, BMMSCs at a dose of 13 million cells were embedded in collagen gel and transplanted into the cartilage defect and covered with autologous periosteum. The clinical improvement was not significantly different from the control group, but the arthroscopic and histological evidence was better in the transplanted group than the control arm. Since then many studies have been published, but still many contentious issues regarding cell therapy in OA are being discussed. We will try to discuss a few burning issues in

**a.Level of evidence regarding the use of MSC therapy in OA**: Jevotovsky et al. [106] did a systemic review of 61 studies to look at the study evidence level, MSC protocol, treatment results, and AEs. The levels of evidence were defined by Marx et al. stating the level of evidence as level 1, randomized controlled trial; level II, prospective cohort study or observational study with dramatic effects; level III, retrospective cohort study or case–control study; level IV, case series; and level V, mechanism-based reasoning [107]. These levels of evidence help physicians to come to clinical decisions. In this review, a total of 2390 patients in 61 studies was identified. Most of the studies used adipose-derived stem cells (ADSCs) (n = 29) or bone marrow-derived stem cells (BMSCs) (n = 30). The majority of the studies (57%) were level IV evidence which consists of therapeutic case series without comparative groups. Only five and nine studies were level I and level II evidence, respectively, in a total of 288 patients. Additionally, 11% were level III retrospective cohort studies, and 8% were level V single-patient case reports. The published data highlights the need for more level I and level II evidence to evaluate the role of MSC treatment in OA patients. However, the majority of the studies have reported positive results and an association between MSC therapy and

symptomatic and radiologic improvement in these patients.

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

**of osteoarthritis of the knee joint**

with a scaffold at the site of cartilage injury.

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

the safety of intra-articular administration of culture-expanded stem cells. A total of 844 procedures (mean follow-up of 21 months) was analyzed. Four SAEs were reported—one infection following bone marrow aspiration (BMA) that resolved with antibiotics, one pulmonary embolism after 2 weeks of BMA, and two adverse events not related to the therapy. Other adverse events documented were increased pain/swelling and dehydration after BMA. In another review, a recent analysis of adverse events (AEs) in 2372 orthopedic patients treated with autologous stem cell therapies and followed up for 2.2 years has been published [70]. The common AEs reported included post-procedure pain and pain due to progressive degenerative joint disease in under 4% of the population. Hence, we can conclude that the systemic administration of MSC including intra-articular administration is safe.

#### **8.2 Efficacy of stem cells including mesenchymal stromal cells in clinical trials of osteoarthritis of the knee joint**

Several clinical trials have been conducted using bone marrow mononuclear cells, adipose tissue-derived stromal vascular fraction (AD-SVF), adipose tissuederived mesenchymal stromal cells (AD-MSCs), or bone marrow-derived mesenchymal stromal cells (BMMSCs) in OA of the knee joint. The list of the published clinical trials in chronological order is given in **Table 2**. Administration of the cells has been fairly standardized, with the cells being administered either directly intraarticularly or under ultrasound guidance. Few trials have been conducted using the arthroscopic method of administration with direct implantation of the cells alone or with a scaffold at the site of cartilage injury.

The first clinical study has been published way back in 2002 by Wakitani et al. [71]. In this study of 12 patients who underwent high tibial osteotomy, BMMSCs at a dose of 13 million cells were embedded in collagen gel and transplanted into the cartilage defect and covered with autologous periosteum. The clinical improvement was not significantly different from the control group, but the arthroscopic and histological evidence was better in the transplanted group than the control arm. Since then many studies have been published, but still many contentious issues regarding cell therapy in OA are being discussed. We will try to discuss a few burning issues in this chapter:

**a.Level of evidence regarding the use of MSC therapy in OA**: Jevotovsky et al. [106] did a systemic review of 61 studies to look at the study evidence level, MSC protocol, treatment results, and AEs. The levels of evidence were defined by Marx et al. stating the level of evidence as level 1, randomized controlled trial; level II, prospective cohort study or observational study with dramatic effects; level III, retrospective cohort study or case–control study; level IV, case series; and level V, mechanism-based reasoning [107]. These levels of evidence help physicians to come to clinical decisions. In this review, a total of 2390 patients in 61 studies was identified. Most of the studies used adipose-derived stem cells (ADSCs) (n = 29) or bone marrow-derived stem cells (BMSCs) (n = 30). The majority of the studies (57%) were level IV evidence which consists of therapeutic case series without comparative groups. Only five and nine studies were level I and level II evidence, respectively, in a total of 288 patients. Additionally, 11% were level III retrospective cohort studies, and 8% were level V single-patient case reports. The published data highlights the need for more level I and level II evidence to evaluate the role of MSC treatment in OA patients. However, the majority of the studies have reported positive results and an association between MSC therapy and symptomatic and radiologic improvement in these patients.

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

compared to the HA alone and disease control groups [43].

Stempeucel® doses of 0.65 × 106

(50 × 106

various methods (**Table 1**).

phase II clinical trial in Indian patients [43].

**8. Clinical trials in osteoarthritis of the knee joint**

**8.1 Safety of mesenchymal stromal cells in clinical trials**

1.3 × 106

OA model in Wistar rats. We demonstrated the dose-dependent efficacy of two

significant dose-dependent reduction in pain scores was observed in both low and high Stempeucel® doses compared to the HA alone and disease control group. Histological evaluation of joint tissue sections in all study groups showed significant improvement in proteoglycan staining in both low and high Stempeucel® administered groups indicating significant regeneration of the cartilage in both groups

Similar to the animal model we used, other scientific groups have created articular cartilage defects in small animals, such as mice [52], rats [43, 53, 54], and rabbits [55, 56]. Smaller animal models are cost-effective and easy to house, and rodents are available in a variety of genetically modified strains with minimal biological variability [57, 58]. However, the small joint size, thin cartilage, altered biomechanics, and increased spontaneous intrinsic healing hamper the study of the regenerative capacity of stem cells and these mechanisms of healing which cannot be fully extrapolated to human cartilage repair [59, 60]. Rodents have mainly been used to assess the chondrogenesis of cell-based therapies by subcutaneous, intramuscular, and intra-articular implantations of cells [60]. Of all small animals, the rabbit model is the most utilized model in cartilage regeneration studies because of the slightly larger knee joint size than rodents [55, 56]. Despite their limited translational capacity, small animals can be very useful as a proof-of-principle study and to assess therapy safety before moving on to preclinical studies using larger animals [60]. Large animal models play a more substantial role in translational research because of a larger joint size and thicker cartilage; however, their preclinical use is often hindered by high costs and difficulties in animal handling. A variety of large animal models have been used to investigate cartilage repair strategies, including horses [61], dogs [62], sheep [63, 64], goats [65], and pigs [66], each with their own strengths and limitations. We have listed some relevant published studies which have used autologous, allogeneic, or xenogeneic BMMSCs to treat OA induced by

Based on the positive efficacy outcomes of our preclinical study, subsequently, we demonstrated the safety and optimal dose for efficacy in a phBMMSC product, Stempeucel®, in a randomized, double-blind, placebo-controlled dose-finding

Lalu MM et al. conducted a systematic review of clinical trials that examined the use of MSCs to evaluate their safety [68]. A total of 36 studies having 1012 participants with different clinical conditions was evaluated. Eight studies were randomized control trials (RCTs) and enrolled 321 participants. Only prospective clinical trials that used the intravenous or intra-arterial route of administration in different age groups were analyzed. Meta-analysis did not detect an association with MSC administration and acute infusional toxicity, organ system complications, infection, and death. There was a significant association between MSCs and transient fever at or shortly after MSC administration which was not associated with long-term sequelae. Most importantly, the meta-analysis showed no serious adverse event due to the administration of MSCs and specifically found no association between MSCs and tumor formation. In another study, Peeters et al. [69] did a systemic review of

HED) followed by an injection of hyaluronic acid (HA). A

human equivalent dose, HED) and

(25 × 106

**58**


**61**

**Author/ year** Koh et al. (2012)

Orozco et al. (2013) Koh et al. (2013)

Van

21

Case series

KL grade 2 or

Auto SVF

Nil

grade 3

Pham

et al.

(2014)

Koh et al.

37 knees

Case series

KL grade 1 or

Auto AD-MSC, 3.8 × 106

Nil

IKDC score, Tegner

IKDC and Tegner activity scale

26.5 months

[81]

scores significantly improved,

ICRS grades showed 2 of the

37 lesions (5%) were grade I

(normal) and 7 (19%) were

grade II (near normal)

Significant improvement in

24 months

[82]

all clinical outcomes, 87.5% of

patients (14/16) improved or

maintained cartilage status

activity scale, and

cartilage repair

assessed using ICRS

grading

grade 2

cells

(2014)

(35

patients)

Koh et al.

30

Case series

KL grade 2 or

Auto SVF + PRP, 42 × 106

Nil

Lysholm score,

KOOS, VAS score,

arthroscopic

evaluation (n = 16)

grade 3

cells

(2015)

18

Case series

KL grade 3 or grade 4

Auto AD-MSC, 1.18 × 106

cells + PRP

Nil

12

Case series

KL grade 2 to grade 4

cells

Auto BMMSC, 40 × 106

Nil

VAS score, Lequesne indexes, WOMAC scores, MRI T2 mapping

All clinical scores decreased significantly, MRI T2 mapping showed improvement of cartilage quality

WOMAC, Lysholm, Tegner,

26 months

[79]

and VAS scores improved

significantly, WORMS score in

MRI improved significantly

WOMAC score, Lysholm score, Tegner activity

scale, and VAS

scores, MRI

(WORMS score)

VAS, Lysholm score,

VAS scores improved, Lysholm

8.5 months

[80]

scores increased, MRI showed

increased cartilage thickness

and MRI

25

Case–control study

KL grade 3 or grade 4

Arthroscopic debridement + Auto AD-MSC, 1.89 × 106 cells + PRP

Arthroscopic debridement + PRP

Lysholm score, Tegner activity scale, and VAS scores

The clinical scores preoperatively were significantly poorer than those of the control group but at the last follow-up visit were similar and not significantly different between the two groups

12 months

[78]

**Sample size**

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up period**

18 months

[77]

**Reference**

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

*DOI: http://dx.doi.org/10.5772/intechopen.86016*


#### *The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

**60**

**Author/**

**Sample** 

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up** 

**Reference**

**period**

16 months

[71]

**year**

Wakitani

24

A single-arm

Stage I to stage

Auto BMMSC, 13 × 106

High tibial

Hospital for Special

No significant difference in

clinical evaluations between

the two groups, arthroscopy

and histology showed partially

hyaline cartilage-like tissue

Decreased VAS pain scores;

6 months

[72]

increased the range of motion;

MRI, statistically significant

cartilage and meniscus growth

Lysholm and RHSSK scores

12 months

[73]

showed statistically significant

improvement, MRI of three

patients revealed complete

defect fill

Surgery knee rating

scale, arthroscopy

and histology

osteotomy

(n = 12)

cells embedded in

soluble collagen (n = 12)

II Ahlback

changes

control study

(2002)

Centeno

1

Case study

—

Auto BMMSC, 2.4 × 106

Nil

VAS, ROM, and

MRI

cells + 1 ml NC + 1 ml

PRP

et al.

(2008)

Haleem

5

Case series

Outerbridge

Auto BMMSC + PR-FG,

Nil

Lysholm and

RHSSK scores and

X-rays and MRI

15 × 106 cells

grade III or

grade IV

et al.

(2010)

Nejadnik

72

Cohort study

Lesion grade 3

Auto BMMSC

n = 12 each in

ICRS, SF-36, IKDC,

SF-36 showed physical role

24 months

[74]

functioning improvement

in the BMMSC group, no

difference in other outcome

measures

Lysholm knee scale

and Tegner activity

level scale

BMMSC and

chondrocyte

group

or grade 4

et al.

(2010)

Davatchi

4

Single-arm

Moderate to

Auto BMMSC, 8–9 × 106

Nil

VAS pain score,

Walking time for the pain to

6 months

[75]

appear improved for three

patient, VAS pain score and

number of stairs to climb

improved for all patients,

improvement in crepitus

time to walk and

number of stairs to

climb to produce

pain, the resting

time to induce the

gelling pain, ROM

and patellae crepitus

Arthroscopy and

Arthroscopy showed articular

26 months

[76]

cartilage regeneration and

histologically showed hyaline

cartilage

histology

severe OA

cells

study

et al.

(2011)

Saw et al.

5

Single-arm

ICRS grade 3

Auto PBPC (8 ml) + HA/

Nil

weekly injection × 5

or grade 4

study

(2011)

**size**


**63**

**Author/ year** Pak et al. (2016)

Pers et al.

18

Open-label,

KL grade 3 or

Auto AD-MSC, low

Nil

Safety, WOMAC, VAS,

PGA, SAS and KOOS

index, MRI, dGEMRIC

in 6 patients

(2 × 106), medium

(10 × 106), and high dose

(50x106), 6 patients each

phase I study

grade 4

(2016)

Koh et al.

80

RCT

ICRS grade 3 or

Auto AD-MSC + fibrin glue

n = 40 (group 2)

Lysholm score,

KOOS, VAS score,

MRI, cartilage repair

tissue scoring system,

arthroscopy and

histology

(microfracture)

+ microfracture, 5 × 106

cells (group 1)

grade 4

(2016)

Gupta

60

Double-blind,

KL grade 2 or

Allo BMMSC + HA, four

n = 20 (placebo

Safety, VAS, ICOAP,

WOMAC, MRI,

WORMS score

+ HA)

doses (25, 50, 75, 150 × 106

cells)

phase II, RCT

grade 3

et al.

(2016)

Lamo-Espinosa

30

Phase I/phase

KL grade 2 to

Auto BMMSC + HA, two

n = 10, (HA)

Safety, VAS score,

WOMAC, MRI,

WORMS

doses (10 and 100 × 106

cells)

II, RCT

grade 4

et al.

(2016)

de Windt

10

Phase I/phase

Modified

Allo BMMSCs +10 or 20%

Nil

Safety, KOOS, VAS,

MRI, second-look

arthroscopy, histology

autologous chondrons

II single-center

Outerbridge

grade 3 or

grade 4

study

et al.

(2017)

3

Case reports

KL grade 3

Auto AD-SVF + HA + PRP,

Nil

FRI, ROM and VAS

All clinical scores improved in

4.5 months

[89]

three patients, MRI showed

cartilage-like tissue regeneration

Safety established, low dose

6 months

[90]

most effective, and all parameters

improved as compared to baseline,

dGEMRIC improved in three patients

MRI, better signal intensity for

24 months

[91]

repair tissue in group 1 (80%) as

compared to 72.5% in group 2;

KOOS pain and symptom

subscores, significantly greater

for group 1; arthroscopy and

histology, no significant difference

Safety established; AE were

24 months

[43]

predominant in the higher-dose

groups; VAS, ICOAP, and WOMAC

scores best in the lowest dose; MRI,

no significant difference

Safety established. VAS,

12 months

[92]

WOMAC, and WORMS scores

significant in high-dose group at

12 months follow-up

No SUSAR; KOOS and VAS scores

12 months

[93]

improved significantly; MRI,

showed complete filling of the

defect; arthroscopy, effective

defect fill, and integration in the

surrounding tissue; histology,

positive staining for both type I and

type II collagen and proteoglycan

score, MRI

PRP repeated weekly × 3

**Sample size**

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up period**

**Reference**

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

*DOI: http://dx.doi.org/10.5772/intechopen.86016*


#### *The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

**62**

**Author/**

**Sample** 

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up** 

**Reference**

**period**

12 months

[83]

**year**

Munar

50

Case series

KL grade 2 to

Auto BMMSC, 40 × 106

Nil

VAS, Lequesne and

All clinical scores improved,

T2 mapping, PCI decreased

WOMAC indices,

MRI (T2 mapping)

VAS pain score,

time to walk and

number of stairs to

climb to produce

pain, the resting

time to induce the

gelling pain, ROM

and patellae crepitus

significantly

All parameters still better than

60 months

[84]

baseline at 5 years follow-up

for three patients

grade 4

cells

et al.

(2015)

Davatchi

4

Case series

Moderate to

Auto BMMSC, 8–9 × 106

Nil

severe OA

cells

et al.

(2016)

Soler

15

Single-arm,

KL grade 2 or

Auto BMMSC, 41 × 106

Nil

VAS score,

The clinical scores improved,

12 months

[85]

SF-36 showed improvement

of parameters, T2 mapping

showed signs of cartilage

questionnaire,

QOL SF-36

questionnaire,

Lequesne functional

regeneration

index and WOMAC

score, MRI (T2

mapping)

VAS score, patient

VAS score and patient

4.8 months

[86]

satisfaction score improved in

all patients

satisfaction scale

open-label

grade 3

cells

phase I/phase

II trial

et al.

(2016)

Sampson

125

Retrospective

KL grade 3 or

Auto BMC + PRP

Nil

(8 weeks apart)

case series

grade 4

et al.

(2016)

Fodor

6 patients

Case series

KL grade 1 to

Auto SVF, 14.1 × 106 cells

Nil

VAS score, WOMAC

VAS and WOMAC scores

12 months

[87]

significantly improved,

ROM and TUG improved,

MRI showed no detectable

structural differences

Clinical outcomes significantly

24 months

[88]

improved, MOAKS and

MOCART score significantly

score, ROM, TUG

test, and MRI

grade 3

(8 knees)

and

Paulseth

(2016)

Kim et al.

20

Case series

KL grade 1 or

Auto AD-MSC, 4.4 × 106

Nil

IKDC score, Tegner

activity scale,

MRI MOAKS and

MOCART score

improved

grade 2

cells

(2016)

patients

(24 knees)

**size**


**65**

**Author/ year**

Russo et al. (2017) Yokota et al. (2017) Jo et al. (2017)

Garay

61

Open-label,

Knee OA

Cell group, BM stimulation

Control

group, oral

scores

acetaminophen

(n = 31)

4 patients,

Safety, WOMAC,

All patients experienced at least

12 months

[103]

one TEAE; VAS and WOMAC,

statistically significant withingroup reduction from baseline

VAS, AQoL-4D,

biomarkers (urine,

C2C and CTX-II;

serum, MIF and

in PRG group, no statistically

significant differences at any

time point between placebo and

PRG groups; MRI, no decrease

in lateral tibial cartilage

volume while the placebo

group showed a statistically

significant cartilage loss

CTX-I; MRI,

MOAKS score)

placebo

administered

with subcutaneous

administration of G-CSF

(n = 30)

phase I/phase

II controlled

trial

Mendoza

et al.

(2017)

Kuah

20

RCT,

KL grade 1 to

Randomized 4:1;

Progenza (PRG) (Allo

AD-MSC + culture

supernatant); 2 groups,

8 pts. each, 3.9 or 6.7

million cells

double-blind,

grade 3

placebocontrolled

et al.

(2018)

18

Single-arm study

Knee OA

Auto AD-MSC; 3 doses (10 × 106, 50 × 106, and 100 × 106 AD-MSCs)

Nil

WOMAC, KSS, KOOS, VAS, MRI, size and depth of the cartilage defect, the signal intensity of regenerated cartilage

and cartilage volume

VAS and WOMAC

BM-SC group showed

6 months

[102]

significant improvement in

knee pain and quality of life

13

Single-arm study

KL grade 3 or grade 4

IA Auto AD- SVF; 2.5 ml SVF containing 3 × 107

SVF cells/knee

Nil

VAS, JKOM, WOMAC

30

Single-arm study

Auto microfragmented adipose tissue

Nil

VAS, KOOS, IKCD, subjective, Tegner Lysholm knee

IKDC and KOOS, improvement of 20 points; VAS and Tegner Lysholm score, improvement in 24 and 31 points, respectively

Scores improved by an average of 35% over baseline for JKOM, 32% improvement in WOMAC, and 40% for pain (VAS)

No TEAE; WOMAC, KSS,

24 months

[101]

and KOOS, improved knee

function; VAS, improved pain

(statistical significance in high

dose); MRI, improvements in

all parameters

KL grade 1 to grade 3, grade > II (ICRS classification)

**Sample size**

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up period**

12 months

[99]

**Reference**

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

6 months

[100]


#### *The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

*Update on Mesenchymal and Induced Pluripotent Stem Cells*

**64**

**Author/**

**Sample** 

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up** 

**Reference**

**period**

12 months

[94]

**year**

Turajane

60

RCT, singlecenter study

KL grade 1 to grade 3

3 groups, 20 each; first group (AAPBSC + PRP

20 patients, IA,

Avoidance of TKA

TKA done in three patients

in the control group but not

in the cell group; WOMAC,

all groups reached statistically

significant improvements

within the individual (intra)

groups

intervention and

WOMAC scores

HA alone

+ G-CSF+ HA + MCS);

second group (AAPBSC

+ PRP + HA + MCS); third

group (control) (all given

weekly × 3 injections)

et al.

(2017)

Shapiro

25

RCT,

Bilateral OA,

25 knees; 5 ml of

25 knees; sterile

ICOAP, VAS scores,

No SAE, patients had a

6 months

[95]

similar decrease in scores in

VAS and ICOAP scores in

both BMAC- and salinetreated arthritic knees

MRI, T2 mapping

saline, 15 ml

Auto BMAC +10 ml

of platelet-poor bone

marrow plasma

KL grade 1 to

et al.

patients,

single-blind,

50 knees

placebocontrolled

grade 3

(2017)

Park

7

Open-label,

KL grade 3 and

Two doses; Allo hUCB

Nil

ICRS cartilage

VAS and IKDC improved at

7 years

[96]

24 weeks and stable till 7 years;

histology at 1 year showed

hyaline-like cartilage; MRI at

3 years showed the persistence

of regenerated cartilage

Safety established, WOMAC

18 months

[97]

scores not significant between

two arms at 6 and 12 months

but significant at 18 months;

increased Lysholm and VAS

scores in the treatment group

compared with the placebo;

MRI, MRI demonstrated

cartilage layer was thicker in

the treatment group

WOMAC scores significantly

12 months

[98]

lower in treatment arm than

baseline; MRI, no change

repair, VAS, IKDC,

MRI, histological

findings

MSCs and HA hydrogel

ICRS grade 4

single-arm,

phase I/

phase II

et al.

(2017)

Nguyen

30

Placebocontrolled

KL grade 2 or

15 patients; AM + Auto

15 patients, AM

Safety, WOMAC,

Lysholm, and

modified VAS

scores, MRI

AD (SVF + PRP; 107 SVF

cells/ml)

grade 3

et al.

(2017)

Pintat

19

Single-arm

Patellofemoral

IA AD-MSC + PRP

Nil

WOMAC, MRI, T2

mapping

study

OA

et al.

(2017)

trial

**size**


**67**

dose (2 × 106

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

**b.Best source of MSC for treatment of OA**: Many studies have been published using different sources of MSCs, and there is no consensus as to which MSC type is the most effective in treating OA. Recently few studies have been published using SVF, bone marrow aspirate concentrate, and micro-fragmented adipose tissue, which further adds to the variability of this issue. The most common problem affecting the clinical outcome in OA is the tendency of MSCs to differentiate into fibrous-like tissue instead of hyaline cartilage [108]. To eliminate or reduce chondrogenesis of the injected MSCs, one school of thought is to identify new sources of MSCs for cartilage repair. Recently synoviumderived stem cells have been used for OA study as it is believed that epigenetic memory may play a role and impact the specific lineage differentiation of MSCs [109]. Hence, the use of synovium stem cells predicts a better outcome as chondrogenic differentiation is expected as it belongs to the same lineage. Fetal stem cells have higher plasticity and proliferation ability than adult stem cells. Hence, fetal tissue-derived stem cells, especially derived from the fetal cartilage, may show higher chondrogenic activity [110] and may be the ideal source of cells for OA. More controlled clinical trials are required to come to a conclusion as to which cell type may be the best choice for the effective treatment of OA.

**c.Autologous or allogeneic source of MSCs**: Most of the published trials used autologous MSCs to minimize immune response, which may lead to best clinical outcomes. Six of the studies in **Table 2** attempted to investigate the potential application of allogeneic MSCs [43, 48, 93, 96, 103, 105] in OA. Recently in the last 2–3 years, most of the studies have attempted to use an allogeneic source of MSCs due to the ease of application. Further, no observed serious adverse effects indicate the safety of allogeneic cells in OA. Around 3000 patients have been administered allogeneic MSCs for different conditions, and no immune response has been reported to date [111]. In a recently published trial using allogeneic umbilical cord-derived MSCs (UC-MSC) in knee OA, patients were randomized to receive hyaluronic acid at baseline and 6 months

No serious adverse events were reported. At 12 months of follow-up, MSC-2 treated group had significantly lower levels of pain [visual analog score (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), total score, and pain subscale] than HA group [48]. Hence, it can be safely concluded that the use of allogeneic MSCs is safe and may be efficacious in OA.

**d.The optimal dose of MSCs for best efficacy in OA**: MSCs have been used in different doses in several clinical trials of OA (**Table 2**). The dose varied from as low as 1.18 million cells [79] to as high as 150 million cells [43]. In a study by Koh et al. [79], 18 patients were given intra-articular injections with adipose tissuederived MSCs in a mean dose of 1.18 million cells and platelet-rich plasma. At 26 months of follow-up, patients had significant improvement in VAS, Lysholm, and WOMAC scores. Magnetic resonance imaging (MRI) was evaluated using WORMS score and showed statistically significant improvement in the total and cartilage scores. In another dose-finding study, Pers et al. [90] recruited 18 patients who were treated with autologous AD-MSCs in three different doses: low

After 6 months of follow-up, the procedure was found to be safe, and no serious adverse events were reported. Patients in the low dose had significant improvement in pain levels and functions as compared to baseline. In a dose-finding study conducted by Gupta et al. [43], four different doses (25, 50, 75 and 150 million

) UC-MSC at baseline (MSC-1, *n* = 9), or

cells), and high dose (50 × 106

× 2; MSC-2, n = 9).

cells).

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

(HA, *n* = 8), single-dose (20 × 106

repeated UC-MSC doses at baseline and 6 months (20 × 106

cells), medium dose (10 × 106

*patient global assessment; PR-FG, platelet-rich fibrin glue; PRP, platelet-rich plasma; QOL, quality of life; RCT, randomized controlled trial; RHSSK, Revised Hospital for Special Surgery knee scores; ROM,* 

*range of motion; SAE, serious adverse event; SAS, Short Arthritis Assessment Scale; SF-36, Short Form-36 quality of life questionnaire; SUSAR, suspected unexpected serious adverse reaction; SVF, stromal* 

*vascular fraction; TEAE, treatment emergent adverse event; TKA, total knee arthroplasty; TUG, timed up and go; UC-MSC, umbilical cord-derived MSC; VAS, visual analog pain score; WOMAC, Western* 

*Ontario and McMaster Universities Osteoarthritis Index; WORMS, whole-organ magnetic resonance imaging score.*

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*


*Update on Mesenchymal and Induced Pluripotent Stem Cells*

**66**

**Author/**

**Sample** 

**Study design**

**Grade of OA**

**Cell type and dose**

**Control group**

**Outcome measures**

**Outcomes**

**Follow-up** 

**Reference**

**period**

12 months

[48]

**year**

Matas

29

Phase I/phase

KL grade 1 to

Allo UC-MSC, single

9 patients, HA

VAS, WOMAC,

No SAEs, repeat dose group

had a significant decrease in

VAS and WOMAC scores as

compared to HA group, no

changes in function subscale,

SF36, and MRI

WOMAC, significant

6 months

[104]

improvements in total score,

pain, and physical function

subscales and improvement

in painless walking distance

compared with placebo

No SAEs; significant knee ROM

24 weeks

[105]

improvement at 2 and 24 weeks;

VAS, no change; KOOS,

improvement till 8 weeks; MRI,

chondral thickness improved

in about 10% of the total knee

joint area AT 24 weeks

*AAPBSC, autologous activated peripheral blood stem cells; AD-MSCs, adipose tissue-derived mesenchymal stromal cells; AEs, adverse events; Auto, autologous; Allo, allogeneic; AM, arthroscopic microfracture;* 

*AQoL-4D, assessment of quality of life 4D questionnaire; BMAC, bone marrow aspirate concentrate; BMMSCs, bone marrow-derived mesenchymal stromal cells; C2C, type II collagen C2C peptide; CTX-1,* 

*C-terminal telopeptide of type I collagen; CTX-II, C-terminal telopeptide of type II collagen; dGEMRIC, delayed gadolinium-enhanced magnetic resonance imaging of cartilage; FRI, functional rating index;* 

*HA, hyaluronic acid; G-CSF, granulocyte colony-stimulating factor; IA, intra-articular; ICOAP, Intermittent and Constant Osteoarthritis Pain; ICRS, International Cartilage Repair Society Cartilage Injury* 

*Evaluation Package; IKDC, International Knee Documentation Committee Subjective Knee Evaluation Form; JKOM, Japanese Knee Osteoarthritis Measure; KL grade, Kellgren and Lawrence grade; KOOS,* 

*Knee Injury and Osteoarthritis Outcome Scores; KSS, Knee Society clinical rating system; MIF, macrophage migration inhibitory factor; MOAKS, MRI Osteoarthritis Knee Score; MOCART, magnetic resonance* 

*observation of cartilage repair tissue; MCS, mesenchymal cell stimulation; MRI, magnetic resonance imaging; NC, nucleated cells; PCI, poor cartilage index; PBPC, peripheral blood progenitor cells; PGA,* 

*patient global assessment; PR-FG, platelet-rich fibrin glue; PRP, platelet-rich plasma; QOL, quality of life; RCT, randomized controlled trial; RHSSK, Revised Hospital for Special Surgery knee scores; ROM,* 

*range of motion; SAE, serious adverse event; SAS, Short Arthritis Assessment Scale; SF-36, Short Form-36 quality of life questionnaire; SUSAR, suspected unexpected serious adverse reaction; SVF, stromal* 

*vascular fraction; TEAE, treatment emergent adverse event; TKA, total knee arthroplasty; TUG, timed up and go; UC-MSC, umbilical cord-derived MSC; VAS, visual analog pain score; WOMAC, Western* 

*Ontario and McMaster Universities Osteoarthritis Index; WORMS, whole-organ magnetic resonance imaging score.*

**Table 2.**

*Chronological list of publications of stem cell application for cartilage repair.*

MRI, WORMS score

(baseline and

6 months)

(20 × 106) or repeat dose

(20 × 106 baseline and

6 months), 10 pts. each

II RCT, tripleblind trial

grade 3

et al.

(2018)

Emadedin

43

RCT, phase

KL grade 2 to

Auto BMMSC, 40 × 106

5 ml normal

VAS, WOMAC,

walking distance,

painless walking

distance, standing

time and knee

flexion compared

VAS, KOOS, knee

flexion range of

motion (ROM),

MRI

saline (placebo)

(n = 24)

I/phase II,

grade 4

cells (n = 19)

placebocontrolled, triple-blind

Khalifeh

20

RCT,

KL grade 2 to

Placental-derived MSC,

Normal saline

(n = 10)

50–60 x 106 cells (n = 10)

double-blind,

grade 4

placebocontrolled

Soltani

et al.

(2019)

et al.

(2018)

**size**

cells) of allogeneic BMMSCs were used in a total of 60 patients. At 1 year of follow-up, the lower doses of 25 million had shown improvement in pain levels and function as compared to placebo and baseline. However in a study by Jo et al. [101], 18 patients were injected with autologous AD-MSCs in three different doses: 10, 50, and 100 million cells. At 2 years of follow-up, significant improvement in the Knee Society clinical rating system (KSS), Knee Injury and Osteoarthritis Outcome Score (KOOS), and VAS scores was seen in the highest dose of 100 million cells. As can be seen, most of the studies are single-arm studies without any control arm. Hence, to determine the most efficacious dose in OA, more randomized controlled, dose-finding clinical trials are required.

**e.Selection of endpoints for the conduct of clinical trial**: The FDA 2018 draft guidance document for OA regarding the development of structural endpoints for the development of drugs, devices, and biological products for treatment states that approvals for OA to date have been based on patient-reported outcome measures that assess pain and function. For the development of new product in OA, the goal of treatment should be inhibition of structural damage or targeting the underlying pathophysiology associated with OA or significantly delay the complications of joint failure and the need for joint replacement and also to reduce the deterioration of function and worsening of pain. All of the above may be taken into consideration for the development of endpoints for the study in OA [112].

Recently a meta-analysis was done to evaluate the different endpoints used to see the therapeutic efficacy and safety of MSCs for the treatment of patients with knee osteoarthritis [113]. Five hundred eighty-two patients in 11 randomized controlled trials were included in this meta-analysis. It showed that MSC treatment significantly improved VAS and International Knee Documentation Committee (IKDC) scores after 24 months of follow-up compared to controls. MSC therapy also showed significant improvement in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lequesne algofunctional indices (Lequesne), Lysholm knee scale (Lysholm), and Tegner activity scale (Tegner) at 12 or 24 months of follow-up. Hence, all the endpoints used currently for evaluation of efficacy in OA have shown significant improvement in different clinical trials:

**f. MRI to evaluate cartilage regeneration**: MRI has emerged as the leading method of imaging soft tissue structures around joints. An ideal MRI study for the cartilage should provide an accurate assessment of cartilage thickness and volume, show morphologic changes of the cartilage surface, show internal cartilage signal changes, and allow evaluation of the subchondral bone for signal abnormalities. Also, it would be desirable for MRI to provide an evaluation of the underlying cartilage physiology, including providing information about the status of the glycosaminoglycan (GAG) and collagen matrices [114]. But, in actual, there is an absence of a standard system by MRI to evaluate cartilage regeneration. Many studies as given in **Table 2** that have used MRI to evaluate cartilage regeneration are only qualitative. It is recommended to use validated imaging outcomes for cartilage regeneration for scientifically validating cell-based therapies, thus advancing the field. The most common parameters used for evaluation of cartilage regeneration by MRI are cartilage thickness in different points in all the compartments of the joint [97], cartilage volume [101], whole-organ magnetic resonance imaging score (WORMS) [43, 48], T2 relaxation time mapping [78, 83, 85, 95, 98], MRI Osteoarthritis Knee Score (MOAKS) score [88, 103], magnetic resonance observation of cartilage repair tissue (MOCART) score [88],

**69**

**8.3 Clinical trials in India**

severe adverse events or complications reported.

*The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee*

and contrast-enhanced imaging technique known as delayed gadoliniumenhanced MRI of cartilage (dGEMRIC) [90]. Among all the parameters, T2 mapping and WORMS seem to be the most commonly used qualitative parameters used for evaluation of cartilage regeneration as it is sensitive to both changes in cartilage hydration and collagen fibril orientation. In a study by Orozco et al. [78], T2 relaxation measurements demonstrated a highly significant decrease of poor cartilage areas (on average, 27%), with the improvement of cartilage quality in 11 of the 12 patients. In another study by Rich et al. [83], a total of 50 patients was evaluated by T2 mapping at 12 months of follow-up after administration of autologous BMMSCs. The mean poor cartilage index (PCI) significantly decreased in 37 of 50 patients (74%), 10 remained the same (20%), and 3 worsened between 7 and 10% (6%). Hence, cartilage T2 mapping may be a sensitive marker for monitoring cartilage quality in subjects with knee OA as it allows us to accurately determine the grade of disorganization of the extracellular matrix.

**g.Use of MSC alone or MSC with a scaffold for intra-articular injection in OA**: When MSCs are injected intra-articularly alone, MSCs scatter widely in the joint, making it impossible to obtain consistent local concentration at the site of cartilage defect. Hence, with a hope to enhance their efficacy in cartilage regeneration, MSC implantation using scaffolds is being attempted in different clinical trials so that the cells are delivered to the site of interest. Compared to direct intra-articular injection, MSC delivery via a scaffold affords more control of proliferation, matrix production, and self-renewal which may help in the regeneration/repair of degenerated or damaged articular cartilage. Different scaffolds have been designed as the delivery system for the repair of articular cartilage. The different scaffolds which can be used are either made of poly-lactic-coglycolic acids (PLGA) [115], collagen [116], gelatin [117], tricalcium (TCP) [118], poly-lactic acid (PLA) [115], hyaluronic acid (HA) [119], poly-glycolic acid (PGA), or fibrin glue [120]. HA has been used frequently for implantation of MSCs into the joint. Many clinical studies (**Table 2**) have used HA as scaffold along with MSCs for implantation of the cells. Cartistem®, an approved drug by the Korean FDA for knee OA, is a combination of human umbilical cord blood-derived MSCs and sodium hyaluronate which is directly implanted at the site of cartilage injury into the joint by arthroscopy [96, 121]. Hence, cells with scaffold are the ideal combination for intra-articular delivery for cartilage degeneration. However, further studies are necessary to find optimal implanta-

tion vehicles that can result in the regeneration of articular cartilage.

Few clinical trials using autologous or allogeneic MSCs or mononuclear stem cells in OA have been conducted in India. The trials registered in the Clinical Trials Registry of India are the two trials done by Stempeutics (one phase II trial completed and the other phase III trial ongoing). However, one published trial by Bansal et al. [122] for the single-arm study was done in India in which a total of 10 patients were treated with AD-MSCs. The patients were evaluated for safety, WOMAC, 6-minute walk test (6MWT), and MRI for cartilage thickness. The patients were followed up for 2 years. The total WOMAC and its subscale scores and 6MWT were significantly improved at all-time points till 2 years of follow-up. Cartilage thickness as determined by MRI improved by at least 0.2 mm in six patients, was unchanged in two patients, and decreased by at least 0.2 mm in two patients. The authors concluded that the procedure demonstrated a strong safety profile with no

*DOI: http://dx.doi.org/10.5772/intechopen.86016*

#### *The Role of Mesenchymal Stromal Cells in the Management of Osteoarthritis of the Knee DOI: http://dx.doi.org/10.5772/intechopen.86016*

and contrast-enhanced imaging technique known as delayed gadoliniumenhanced MRI of cartilage (dGEMRIC) [90]. Among all the parameters, T2 mapping and WORMS seem to be the most commonly used qualitative parameters used for evaluation of cartilage regeneration as it is sensitive to both changes in cartilage hydration and collagen fibril orientation. In a study by Orozco et al. [78], T2 relaxation measurements demonstrated a highly significant decrease of poor cartilage areas (on average, 27%), with the improvement of cartilage quality in 11 of the 12 patients. In another study by Rich et al. [83], a total of 50 patients was evaluated by T2 mapping at 12 months of follow-up after administration of autologous BMMSCs. The mean poor cartilage index (PCI) significantly decreased in 37 of 50 patients (74%), 10 remained the same (20%), and 3 worsened between 7 and 10% (6%). Hence, cartilage T2 mapping may be a sensitive marker for monitoring cartilage quality in subjects with knee OA as it allows us to accurately determine the grade of disorganization of the extracellular matrix.
