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

Osteoarthritis (OA) is one of the most common chronic, inflammatory, and degenerative diseases affecting the synovial joints, the hip, and the knee. OA is commonly managed clinically by treating pain with anti-inflammatory medicines using nonsteroidal anti-inflammatory drugs (NSAIDS) or analgesics. In severe OA patients, invasive knee replacement surgery is the last option. Treatment of OA using mesenchymal stromal cells (MSCs) has been widely explored due to their anti-inflammatory properties and chondrogenic differentiation potential. In this chapter, we comprehensively discuss in detail the in vitro OA potency development, OA preclinical studies, and clinical trials conducted using MSCs.


Introduction
Common factors linked to osteoarthritis (OA) occurrence are increasing age (>55 years) and obesity [1]. The gender also seems to play a major role, where the majority of OA patients are women and higher prevalence has been liked to menopause. Radiological evidence suggests that about 70% of women above the age of 65 years are affected by OA [2,3]. Other factors such as genetic predisposition, extrinsic environmental factors, nutrition, and lack of exercise are reasons for the increased prevalence of OA. It has been reported by the World Health Organization (WHO) that 10-15% of the populations aged >60 years exhibit a certain degree of OA [4]. It has been reported by the National Health Portal of India that 22-39% of the Indian population are affected by OA. As reported by the United Nations Organization (UNO), 130 million people will be affected by OA with over 40 million people with severe disability due to disease progression [3].
The etiology of OA is believed to be multifactorial. Some of the main reasons include the biomechanical disease progression due to the narrowing of space in the joints, bone hypertrophy, and formation of new osteophytes in the articular margins causing stiffness and pain in the joints. In addition, an imbalance in the synthesis and release of cytokines by chondrocytes in the disease state could be the main reason for the continual inflammatory state in the joint. During the initial stages of OA, catabolic interleukins (IL) such as IL-1α and IL-1β and tumor necrosis factor α (TNFα) increase inflammation affecting cartilage metabolism and homeostasis. TNFα is a proinflammatory cytokine implicated in the degradation of matrix proteins synthesized by

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 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.

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][31][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 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.

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 Update on Mesenchymal and Induced Pluripotent Stem Cells 6 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.

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 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 × 10 6 (25 × 10 6 human equivalent dose, HED) and 1.3 × 10 6 (50 × 10 6 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].

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 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.

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:  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]  , 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 dose (2 × 10 6 cells), medium dose (10 × 10 6 cells), and high dose (50 × 10 6 cells). 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 ( 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 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], 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 implantation vehicles that can result in the regeneration of articular cartilage.

Clinical trials in India
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 severe adverse events or complications reported.

Stempeutics Research experience in osteoarthritis of the knee joint
The off-the-shelf allogeneic, pooled BMMSC product developed by Stempeutics has completed one phase II clinical trial [43] and currently ongoing phase III trial in knee OA. In our completed phase II trial, we included patients of idiopathic OA in grade 2 or 3 of Kellgren and Lawrence radiographic criteria; patients who had self-reported difficulty in at least one of the following activities attributed to knee pain, lifting and carrying groceries, walking 400 m, getting in and out of a chair, or going up and down stairs; and patients who had been on stable medication, including nonsteroidal anti-inflammatory drugs/opioid analgesics for the past 3 months and in the age group of 40-70 years. All the criteria have to be present before being included in the study [43].

Phase II study in patients with osteoarthritis of the knee joint
The phase II results of Stempeucel® in OA patients have been published [43]. Briefly, it was a double-blind, randomized, placebo-controlled, dose-finding study. In this study, 60 OA patients were randomized to receive different doses of Stempeucel®, 25, 50, 75, and 150 million cells or placebo. Stempeucel® was administered intra-articularly (IA) to the knee joint followed by 2 ml of hyaluronic acid (20 mg). The subjects were followed up for 2 years and were evaluated for safety parameters including AEs, and for efficacy parameters, VAS for pain, Intermittent and Constant Osteoarthritis Pain (ICOAP), WOMAC (total score and its subscales), and MRI were done to evaluate the WORMS score. The intra-articular administration of Stempeucel® was safe with knee pain and swelling as the most common AEs. Clinically relevant improvement in a persistent manner was seen in 25 million dose group in all subjective parameters (VAS, ICOAP, and WOMAC scores) (Figures 2-4). WORMS of MRI knee did not reveal any difference from the baseline and placebo group. It was concluded that intra-articular administration of Stempeucel® is safe and 25 million dose may be the most effective among the doses tested.
Currently, we are conducting a phase III trial in OA of the knee joint. This is a randomized, double-blind, multicentric, placebo-controlled study assessing the efficacy and safety of intra-articular administration of Stempeucel® in patients with osteoarthritis of the knee joint. One hundred and forty-six patients will be  randomized to stem cell and placebo arm in a ratio of 1:1. Seventy-three patients will receive Stempeucel® (25 million) followed by 2 ml of hyaluronan, and 73 patients will receive only intra-articular injection of 2 ml of placebo followed by 2 ml of hyaluronan. The patients will be followed up for a total of 2 years after IMP administration. The details of the study are found in the Clinical Trials Registry of India (CTRI/2018/09/015785).

Conclusion
Osteoarthritis is a common disorder involving damage to synovial joint tissues particularly the cartilage and bone. Current treatments are mostly targeted at end-stage disease, but biological therapies including stem cell therapy show a promise for earlier intervention with a more prolonged benefit. With all the published clinical trial data, it is reasonable to expect that MSCs may prove to be an important therapy for OA. Pooled BMMSCs with their enhanced anti-inflammatory potential, immunomodulatory properties, and secretion of paracrine factors create the optimum environment for a controlled reparative pathway in the affected joint. Pooled BMMSC treatment, perhaps combined with other modalities like a scaffold, would be advantageous in providing treatment in early OA to slow disease progression, thus delaying or avoiding total joint replacement. 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.