Cell-based therapy in the treatment of musculoskeletal diseases

Introduction

The past 2 decades have witnessed growing insights into innate regenerative capacity of mammalian adult tissues and the underlying biology of stem cell niches that govern endogenous repair and rejuvenation. The regenerative capacity of tissues and organs ranges substantially from continuous renewal (eg, hair follicles and gut) to extremely limited repair capacity (eg, heart and CNS). As limited tissues have the capacity to regenerate to a completely healthy tissue following injury, there is intense interest in using exogenous cells and cell-derived products to stimulate endogenous repair pathways. Among these strategies, the use of culture-expanded mesenchymal stem/stromal cells (MSCs) has occupied the attention of many scientists and Orthopedic clinicians resulting in extensive research aimed at defining the potential of these cells and related products for cartilage and bone repair.¹

MSCs are a heterogenous subset of multipotent regenerative cells that can be harvested from various sources including bone marrow (BM), amniotic fluid, placenta, adipose tissue, joint synovium, synovial fluid, dental pulp, endosteum, and periosteum.² MSCs were first described by Friedenstein in the late 1960s to early 1970s as a small population of BM cells characterized by osteogenic potential, adherence to plastic, and the ability to form new, fibroblast-like progeny over many culture passages.^3,4Friedenstein found that these cells could differentiate in vitro not only into osteocytes but also into chondrocytes and adipocytes. Subsequently, Caplan et al in 1991 termed these cells “mesenchymal stem cells” after discovering their ability to differentiate into more than one type of cell that formed connective tissue in many organs and had many different phenotypic endpoints of differentiation. Caplan pioneered the differentiation of the MSCs into bone-forming osteoblasts and cartilage-forming chondrocytes and described the ideal culture medium and conditions for each.⁵

Growing interest in MSCs led to controversy regarding the nomenclature and criteria for defining populations of cells as MSCs. To address this, the International Society for Cellular Therapy (ISCT) published its position specifying the criteria defining the population of MSCs and recommending the use of the name multipotent mesenchymal stromal cells; however, mesenchymal stem cells remain the most popular name. The ISCT’s published criteria described 3 major requirements: (1) cells must be plastic adherent when maintained under standard culture conditions; (2) cells must express cluster of differentiation (CD)73, CD90, and CD 105 markers and should not express CD34, CD45, CD14, human leukocyte antigen complex (HLA)-DR, CD11b or CD19; (3) cells must possess the ability to differentiate into osteoblasts, chondroblasts, and adipocytes in vitro.^6,7

One potential conflict with these criteria is the relationship between MSCs and vascular pericytes. Pericytes are contractile cells that line the outer walls of capillaries and other vascular structures. They have similar cell surface markers and characteristics of MSCs and exhibit many similar properties to MSCs when grown ex vivo.^8-11 For these reasons, some investigators propose that MSCs are derived from pericytes.¹² However, lineage tracing techniques in transgenic mice reveal that pericytes are not present as local progenitor cells in vivo and are not equivalent to MSCs.¹³ Additional clarification derives from the findings of Khosravi et al^14,15 using intra-vital microscopy supporting the idea that tissue-resident mesenchymal progenitor cells are localized between capillaries and represent a distinct population from pericytes. Their work suggests that within bony compartments tissue-resident mesenchymal cells are of perivascular origin and can differentiate into functional pericytes in addition to fibroblasts and osteogenic cells.

Also worth noting is that within the bony compartments are different types of endothelial cell (EC)-lined vessels that are selectively surrounded by a perivascular population of cells that are Runx2 and SP7 positive committed osteoprogenitor cells. Endosteal ECs that strongly express CD31/platelet endothelial cell adhesion molecule-1 and endomucin are termed H-type ECs, which are abundantly surrounded by osteoprogenitor cells. These findings confirm the earlier observations of Aubin et al¹⁶ who described populations of committed and inducible mesenchymal cells in BM. These H-type ECs mediate local growth of vasculature and provide niche signals for perivascular osteoprogenitors, which is important for compromised fracture healing and bone density loss.¹⁷ The concept of perivascular progenitor cells and their H-type EC microenvironment provides an interesting explanation for loss of bone mass density and a possible therapeutic target to enhance osteogenesis and fracture healing.

To conduct this review, we searched for studies using Pubmed and Data.gov including keywords and headings such as “MSCs, stem cells, muscle, articular cartilage, osteoarthritis, meniscus tear, tendon tear, ligament tear, intervertebral disc, fracture, and trauma.” Preference was given to RTCs conducted since 2017. Animal and preclinical studies were included to demonstrate the biological rational and potential mechanisms underlying the use of MSCs.

MSCs application in fracture healing

Fracture healing occurs as a result of a complex sequence of events that often results in complete bone repair to a pre-injury condition. A key feature of fracture healing proceeds through a combination of endochondral and intramembranous ossification.¹² Intramembranous ossification occurs when bone formation ensues without a cartilaginous phase and the majority of cells that contribute are present in the inner osteogenic layer of the periosteum and endosteum. Ossification begins with the condensation of MSCs producing a collagen-proteoglycan matrix that can bind calcium and become mineralized. A BM cavity is formed and calcified bone spicules become surrounded by compact MSCs that form the periosteum and endosteum. Osteogenesis is made possible due to the presence of MSCs that form the inner sheet of the periosteum and the endosteum.^51,52

Endochondral ossification occurs when an initial synthesis of cartilaginous scaffolding is followed by its calcification.⁵³ This calcified scaffold is then resorbed by osteoclasts, which permits vascular invasion and osteogenesis to occur. This process of angiogenesis after a fracture is stimulated by hematoma formation and a subsequent series of intercellular connections and release of growth factors, cytokines, and interleukins that cause chemotaxis to the fracture site. Angiogenesis is crucial for trafficking of MSCs from the systemic circulation and also from previously described perivascular osteoprogenitor cells to the fracture site.^51,54 In addition, H-type ECs mediate local growth of the vasculature and provide a niche signal for perivascular osteoprogenitors.^16,17

Capitalizing on these events, BM extracts have been used for treating nonunion fractures with interest in specific molecular pathways to boost H-type vessel formation and osteogenesis associated with compromised fracture healing or bone mass loss.⁵⁵ Two factors that have been found to act synergistically to promote MSCs migration to fracture sites and fracture site repair are stromal cell-derived factor (SDF)-1 and monocyte chemotactic protein-3.⁵⁶ Others have noted that the expression of bone morphogenic protein-2 and (SDF)-1 increase MSC migration and differentiation to stimulate fracture healing. SDF-1 promotes the autocrine release of VEGF and basic fibroblast growth factor (bFGF) paracrine factor which are both associated with inhibiting cellular apoptosis.⁵⁶ It is important to note that VEGF also plays an essential role in coupling vascular and skeletal morphogenesis. VEGF stimulates the vascular tip as well as bone-forming progenitors along with helping to incite mobilization of nutrition, oxygen, and minerals necessary for osteogenesis. Release of VEGF from MSCs not only plays a role in necessary angiogenesis before osteogenesis but also influences lineage fate between osteoblasts, chondrocytes, or adipocytes.⁵⁷ Also of note, platelets release TGF-β in the inflammatory phase of fracture healing which in turn participates in callus formation and effective chemotaxis of BMD-MSCs.⁵⁸

Understanding the multiple pathways involved in MSC regenerative abilities in bone osteogenesis is important in allowing the design of novel MSC-based therapies to treat fractures. For example, Granero-Moltó et al⁵¹ used a stabilized tibia fracture mouse model to determine the dynamic migration of transplanted MSCs to the fracture site and engraftment within the callus endosteum. In addition, they clarified MSCs’ contribution to the repair process and their role in modulating the injury-related inflammatory response (Figure 1). Building on these concepts, Wang et al⁵⁹ used a C57 murine unilateral, transverse femur fracture model to determine the optimal time to inject BMD-MSCs for fracture healing. Their study had multiple implications: (1) injection of BMD-SCs improved bone healing, (2) injection of BMD-SCs 7 days postfracture promoted accelerated fracture healing and callus/bone quality, (3) the SDF-1-chemokine receptor 4 (CXCR) pathway played a key role in the fracture healing process, and (4) MSCs contributed therapeutically to delayed fracture healing and nonunions. Moreover, Emadedin et al⁶⁰ demonstrated the safety of percutaneous autologous BMD-MSC implantation for the reconstruction of human lower limb long bone atrophic nonunion. However, it was noted that further double-blind, controlled clinical trials are necessary to assess the efficacy of this treatment. Recently, Mott et al⁶¹ preformed a systematic review of 94 studies assessing the evidence for the use of stem cells in fracture healing. The authors found that there was insufficient high-quality evidence to determine the efficacy of stem cells for fracture healing with lack of technique standardization cited as their major limitation. Also, the heterogeneity of assessment of fracture consolidations along with the lack of uniformity in outcome measurements creates significant obstacles to formulating control subjects and measuring outcomes.⁶² To date, there are no registered clinical trials with published data on MSCs and fracture or bone healing. However, in the last 10 years, there are at least 8 clinical trials registered with respect to fracture healing or treatment of nonunion with mesenchymal stem cells. Most trials focus on targeting BMD-MSCs as the primary mesenchymal stem cell treatment for fracture healing (Table 1).^63–70 One trial is randomizing 32 patients, aged 70-85, to treatment with either endomedullary nailing + BMD-MSC treatment or isolated endomedullary nailing. These patients will be followed for 1 year, primarily assessing safety, CT and X-ray, clinical status, and quality of life.⁶⁶ Improvement in clinical status and quality of life for these patients could have a major clinical impact due to the high levels of morbidity and mortality associated with hip fractures in older individuals (Table 1).⁷¹

MSCs application in muscle injury and repair

Following muscle fiber injury, the nuclei of damaged fibers undergo apoptosis. Failure of the replacement of these nuclei partially explains the muscle atrophy and weakness that patients experience after moderate or severe skeletal muscle damage.⁷² The endogenous repair mechanism for this injury derives from skeletal muscle stem cells called satellite cells (also called myosatellite cells), which are activated by injury, migrate to the site of injury, and fuse with injured fibers to repopulate the lost nuclei.⁷² Deploying satellite cells as a therapy represents a possible strategy for the treatment of muscle injuries. However, in vitro expansion of these cells has proven difficult and they exhibit limited post-transplant survival.¹²

As an alternative, BMD-MSCs have been explored as a possible therapeutic strategy. In multiple studies conducted in mice, BMD-MSCs were incorporated into myofibrils of injured muscles and gave rise to Pax7+ satellite cells, a marker for muscle regeneration.^73,74 Furthermore, engrafted MSCs survived repeated cycles of damage without additional transplant with 89% efficiency.⁷⁴ Stromal cell-derived factor 1 SDF-1/CXCR4 signaling and chemokine receptor (CCR2) are implicated in BM cell homing to muscle injury sites.⁷³ A study examining the role of CCR2 on muscle regeneration and BM-derived cells found that mice lacking CCR2 demonstrated fewer BM-derived cells and macrophages along with decreased muscle regeneration.⁷⁵ Klimczak et al⁷⁶ hypothesized that the delivery of MSCs into dystrophic muscle will create a microenvironment/ niche supporting homing of myogenic precursor and enhance tropism of stem cells of myogenic origin with CXCR4+ expression to injured muscle expressing SDF-1. These investigators found that MSCs were viable in injured muscle and contributed to regeneration supporting the importance of microenvironment/niche in MSCs muscle therapy. Iyer et al⁷⁷ also demonstrated that exosomes derived from MSCs facilitate recovery of muscle strain injury in small animal models via reduced expression of TGF-β. They concluded that this resulted from modulation of inflammation, fibrosis, and myogenesis. Large animal model studies demonstrated that MSCs can be used to prevent fatty atrophy that can occur with muscle injury. Flück et al⁷⁸ demonstrated molecular, microscopic, and macroscopic evidence of the prevention of fatty atrophy of detached rotator cuff muscle after tendon repair in an ovine model. They concluded that injection of micro-tissues from MSCs result in a paracrine cascade of events results in expansion of extracellular ground substance, increased tissue water content, and introduction of tenascin C-associated myogenic reactions. Coupled with downregulation of differentiation marker peroxisome proliferator-activated receptor gamma ultimately leads to prevention of fatty atrophy in repaired infraspinatus detachment in sheep models.

At present, there are no clinical trials registered to investigate the effect of MSCs on muscle healing, as all trials focus on the myotendinous junction or the muscle-tendon unit, rather than isolation of the muscle itself. Current studies are also exploring MSC use in the treatment of various dystrophic diseases or spastic palsies.^79–82 However, there is ongoing research investigating the potential role for MSCs in frailty patients to help mitigate sarcopenia, dynapenia and loss of strength with aging. Clinical trials in this area have demonstrated some potential in the CRATUS phase 1 and 2 trials exploring the concept of rejuvenating aged niches and recapitulating their bioactivity using allogenic BMD-MSC in patients with mild frailty. The results of these trials suggest evidence of bioactivity and effectiveness of BMD-MSCs to combat sarcopenia and age-related muscle wasting with improved hand grip strength, increased 6-minute walk distance and reduced circulating TNF-α (inflammatory cytokine linked to frailty related sarcopenia and loss of strength with aging).^83–87 In turn, based upon these promising findings, future clinical trials dedicated to orthopedic muscle injury are warranted to identify treatment targets for muscle strains, traumatic lacerations, and other injuries to muscle belly tissue.

MSCs application in articular cartilage disease and osteoarthritis

An estimated 30.8 million adults in the United States and in excess of 300 million worldwide suffer from osteoarthritis (OA).⁸⁸ Articular cartilage is the hyaline cartilage at the end of long bones and comprises a thin layer bonded to the perforated cortical bone adjacent to intervertebral discs that provides a low friction surface for articulation and transmission of weight between joints.^88,89 Although the half-life of collagen within cartilage is long, articular cartilage heals very slowly and lesions are difficult to manage because of tissue biochemical properties, acellularity, and limited self-renewal potential.^12,88 Moreover, OA is not solely a wear and tear disease but represents a complex process that includes inflammatory and metabolic factors such as metalloproteases and inflammatory cytokines.⁸⁸ In addition, OA affects not just the articular cartilage but the entire joint and surrounding soft tissues particularly the synovium, infrapatellar fat pad, and subchondral bone.⁸⁸

Subchondral bone is the layer of bone beneath the hyaline cartilage and cement line, divided anatomically into the subchondral bone plate and subchondral bone trabeculae. It provides mechanical and nutritional support for cartilage and changes in its microenvironment can impact cartilage metabolism.⁹⁰ The synovium is a specialized tissue lining diarthrodial joints, responsible for maintaining synovial fluid volume and composition to aid in chondrocyte nutrition, and has an outer layer rich in collagen and a relatively acellular inner layer dominated by fibroblasts as well as macrophages. It is important to note that synovial fluid facing macrophages are master regulators of joint homeostasis and play an essential role in the changes that lead to osteoarthritis and other joint inflammation.⁹¹ In osteoarthritis, the synovium undergoes histological changes, characterized by synovial lining hyperplasia, fibrosis, and stromal vascularization, which leads to macrophage infiltration and subsequent cytokine production, angiogenesis, and cartilage degradation.⁹² The secretome of MSCs, which includes growth factors, cytokines, chemokines, and lipids, among others, plays a crucial role in their therapeutic properties. These paracrine factors act on the microenvironment to influence the phenotype of target cells such as promoting anti-inflammatory M2 macrophage polarization.^93,94 Extracellular vesicles (EVs), such as microvesicles or exosomes, are a key part of the secretome and facilitate communication between different cell types and prior in vitro studies suggest immunomodulatory, regenerative, anti-catabolic, and chondroprotective properties.⁹⁵ EVs are an attractive cell-free therapy because they provide the same clinical benefits without the potential risks of live cell infusions. In addition, EVs show promise in the treatment of OA by reactivating aging MSCs; however, the main clinical challenge is the low production of exosomes.⁹³

Given the multitude of tissues affected and the involvement of different inflammatory pathways, the proposed versatility of MSCs have emerged as a potential therapeutic option in the treatment of OA. Several clinical studies have examined the possible benefits of MSCs for OA. A variety of delivery methods and cell processing protocols have been deployed, with encouraging results along with at least one approved product for clinical use.^96–118

MSCs have shown promising clinical benefits in patients with osteochondral defects. Bashir et al⁹⁶ reviewed 4 clinical trials, testing MSCs injection, that demonstrated improved visual analog scale (VAS) pain scores, reduced cartilage defect as seen on magnetic resonance imaging (MRI), and improved functional measures such as functional rating index and ability to climb stairs. A systematic review by Migliorini et al⁹⁷ of MSC injections for knee OA including 18 studies comprising 1069 treated knees tested the impact of treatment on a variety of clinical outcomes, including the VAS, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score, mean walking distance, Lequesne scale, and Knee Injury and Osteoarthritis Outcome (KOOS) score at 6- and 12-month follow-up. This analysis supported the conclusion that MSC injections for knee OA represent a feasible therapy producing a meaningful improvement of all clinical and functional outcomes, regardless of cell source. Most notably, mean walking distance improved from 71.90 to 152.22 and 316.72 m at 6- and 12-month follow-up, respectively. In addition, mean VAS improved from 18.37 to 30.98 and 36.91 at 6- and 12-month follow-up, respectively. However, these results should be taken with caution as many studies do not provide convincing evidence of long-term benefits after 3-5 years.⁹⁸ For example, the meta-analysis performed by Kim et al⁹⁹ examined clinical outcomes of intra-articular injections of MSCs for cartilage repair in knee OA. In their study, they looked at a total of 5 RCTs and found a significant improvement in Lysholm score and WOMAC when MSCs were used in recommended concentrations but no significant difference in cartilage repair when assessed with MRI. They found that despite favorable clinical outcomes there is limited evidence of pain relief and functional improvement in knee OA and that further rigorously conducted, well-powered RCTs with long-term follow-up are required before MSCs can be supported for use for cartilage repair in OA.

In an attempt to provide more clarification on the potential role of MSCs in the treatment of knee OA, Di Zhao et al¹⁰⁰ performed a systematic review and network meta-analysis evaluating the clinical efficacy of intra-articular injection of MSCs. The study evaluated intra-articular injections of hyaluronic acid (HA), leukocyte-poor platelet-rich plasma (LP-PRP), leukocyte-rich PRP (LR-PRP), BMD-MSCs, and AD-MSCs against a saline placebo with 6- and 12-month follow-up. The analysis included 43 studies and looked at VAS scores and WOMAC pain subscores. The results were discussed in comparison to HA injections and demonstrated that at 6 months AD-MSCs provided the most pain relief and that LP-PRP was the most effective for functional improvement. During the 12-month follow-up, both AD-MSCs and LP-PRP showed potential clinical pain relief effects; however, the most effective improvements of functional improvement was achieved with LP-PRP.

Importantly, RCTs continue to emerge supporting the clinical application of MSCs in the treatment of knee OA. A randomized phase 2B placebo-controlled clinical trial conducted that tested the efficacy, safety, and outcome scores of intra-articular injections of autologous AD-MSCs for the treatment of knee OA. AD-MSCs were administered to 12 patients and the group was compared with 12 knees injected with normal saline as a control and were followed for 6 months. The WOMAC score was the primary outcome and with secondary outcomes including radiographic examination and MRI results. The study concluded that a single injection of AD-MSCs led to a significant improvement in the WOMAC score at 6 months compared to the control group. No serious adverse events were observed. MRI evaluation showed no significant changes in cartilage defects at 6 months in the MSC group whereas the defect in the cartilage defects in the control group increased (Figure 2).¹⁰¹

There are multiple registered phase 2/3 clinical trials of note showing promising application of MSC in knee OA (Table 2).^102–115 Of note, 2 clinical trials show promise for improvement in outcomes of pain and/or functional ability after therapy. In addition to the clinical improvements, radiographic assessments of articular cartilage improved with the use of BM-MSCs in the knee.^116,117 One trial followed 32 patients with knee OA over a span of 12 months assessing pain and patient-reported quality of life after treatment with BMD-MSCs, compared to patients randomized to hyaluronate gel treatment. They found the most improvement in the BM-MSC group at 12 months, consistent with other studies.^{12,88,100,106} Additionally, the 5-year follow-up of a multicenter clinical trial with 114 patients with symptomatic, large, full-thickness defects showed superiority of the combination treatment of allogeneic UCB-MSCs and 4% hyaluronate (UCB-MSC-HA) when compared to microfracture with respect to cartilage grading on second look arthroscopy.¹¹⁸

There are currently 2 commercially available products available for use outside of the United States that are evaluated in phase 3 trials. JointStem represents an autologous stem cell-based medication injected into the joint cavity of patients with degenerative arthritis for renewal of cartilage along with alleviation of pain and improved joint function at 9- and 12-month time points. The product is currently approved by the Ministry of Health, Labor and Welfare of Japan and is used in a phase 2b/3a double-blinded multicenter trial scheduled for completion on December 30, 2023.¹⁰⁵ CARTISTEM represents an allogenic UCB-MSC source injected in knee joint cavities and marketed as a treatment of cartilage defects in patients with degrative or repetitive traumatic osteoarthritis. Phase 1/2a clinical trials focusing on the treatment of grade 3-4 articular cartilage defects in knees demonstrated promising IKDC scores at 12 months. The product is currently market-approved for commercial sale by the Ministry of Food & Drug Safety and is currently undergoing phase 3 clinical trial in Japan.¹⁰⁹

In summary, the totality of evidence demonstrates that MSCs, regardless of source, are safe for clinical use to treat knee OA. There have been a number of RCTs demonstrating the potential promising benefits of MSCs with regards to outcome measures such as the WOMAC, VAS, and mean walking distance. However, some studies still demonstrate some inconsistences with regards to clinical pain relief compared to current PRP injections/standards of practice. For example, a recent phase 2/3 four-arm parallel, multicenter, single-blind, RCT with 480 patients demonstrated non-superiority of autologous BM aspirate concentrate, UC tissue-derived MSCs, PRP, and cortical steroid injections in patients with knee OA after 1 year.¹¹⁹ A significant amount of heterogeneity still exists in the literature in regards to outcome, dosing protocols, and dose-dependent effects. In large part, results are short-term demonstrating some improvement over 12 months with few studies showing 5-year follow-up. Important to note that our review preferred more recent studies, for a more comprehensive discussion, Carneiro et al⁹⁸ provides an in-depth review.

MSCs application in meniscus injuries

The knee meniscus functions to transmit joint reactive forces, lubricate, provide nutrition to the cartilage, and act as a shock absorber.¹²⁰ It is a complex tissue comprised of both cells, specialized extracellular matrix molecules, and region-specific innervation and vascularization. In a healthy adult, there are 3 distinct regions of the meniscus: outer vasculature/neural region (red-red zone), inner completely avascular/aneural region (white-white zone), and a region that separates the two called a red-white region which has attributes of both. The vascularization of this tissue is highly relevant to the regenerative potential of the meniscus with the red-red zone having the greatest potential and the white-white zone having the least.¹²¹ Current repair techniques for arthroscopic meniscus repair, namely the inside-out, outside-in, and all-inside techniques are effective in treating lesions located in the peripheral vascularized zone; however, satisfactory treatment of the inner avascular region remains a clinical challenge.^121–123 As a result, there has been a shift toward the utilization of biomaterials and bioengineering for meniscus repair incorporating stem cells and synthetic scaffolds. For example, a study examining the application of nanofiber-based scaffolds seeded with MSCs for the healing of avascular meniscus tears reported that the nanofiber-based scaffolds released collagenase which, enhanced meniscus healing in both in vitro and in vivo settings.¹²³

Recently, several clinical series employing MSCs have been conducted, including 2 by Sekiya et al¹²⁴. The first combined surgical repair and S-MSCs transplantation. This series studied 5 patients with complex degenerative tears of the medial meniscus. They received surgical repair by standard surgical procedure with all-inside and inside-out repairs and a suspension of in vitro cultured autologous S-MSCs were transplanted into the repair utilizing an 18-gauge needle 2 weeks after surgery (Figure 3). Two years following cell transplantation there was an improvement in scores for “pain,” “daily living,” and “sports activities.”¹²⁵ The second study combined meniscus repair with S-MSC transplantation in 6 patients with degenerative flaps and radial tears of the medial meniscus using the same procedure described above. The investigators performed arthroscopy and Lysholm scores along with the appearance of the repair site. The 4 flap tear patients showed tear zones that were completely stable and smooth in 2 patients and partial restoration in 2 patients at 52 weeks. The radial tear patients were reported to have completely healed at the final follow-up as well. The arthroscopy and Lysholm scores at 1 year were found to be higher compared to preoperative levels in all patients. Although the results of these series are encouraging, it is important to note that these studies lacked control subjects.¹²⁶

There is only one randomized double-blind controlled trial studying the effects of MSC injection in knees post partial medial meniscectomy.¹²⁷ The study consisted of 55 subjects in 3 groups receiving percutaneous injections of allogeneic MSCs: 50 × 10⁶ cells, 150 × 10⁶, and a control group only receiving HA. At 12 months, MRI scans demonstrated a significant increase in meniscal volume in 24% of the patients receiving 50 × 10⁶ cells and 6% receiving 150 × 10⁶ cells. None of the control group patients demonstrated increased meniscal volume. However, the study is limited because MRI was the only outcome measure used and clinical outcomes were not assessed.^127–129 Whitehouse et al¹³⁰ published a case series describing 5 patients who received BMD-MSCs injected onto a collagen scaffold and sutured into the white-white zone meniscus tears. At 12 months, 3 of the 5 patients demonstrated significant clinical improvement scores and MRI scans demonstrated in situ repair along with reduced abnormal scaffold signals; however, the other 2 patients had repeat tears and failed treatment at the 15-month follow-up.¹³¹ Despite optimistic results of current human studies, MSC studies in humans with meniscal pathology are still in the early stages and require more studies before a reliable assessment can be made.

With respect to the treatment of meniscal injuries/damage with MSCs, there are currently 3 registered clinical trials in the last 5 years (Table 3).^132–134 The most recent trial investigates the efficacy of SMCS for degenerative meniscal tears.¹³² Another study from 2019 is evaluating the safety of Human Embryonic Stem Cell (hESC)-derived MSC-like cells for a treatment target in meniscus injury.¹³³ The final study is incorporating BMD-MSCs for degenerative meniscal tears. Currently, degenerative disease is the predominant focus of meniscal MSC use, which may translate to traumatic sports injury of the meniscus in the future.¹³⁴

MSCs application in tendon and ligament injuries

Ligament and tendon injuries are common orthopedic problems affecting approximately 17 million patients a year in the United States. These injuries often result in slow healing that requires lengthy rehabilitation and the formation of scar tissue that requires years of remodeling to achieve functional tissue.^135–137 More serious injuries with complete ligament and tendon disruption of require operative repair or tissue augmentation. Multiple approaches have been used in reconstructive surgeries for ligaments and tendons such as allografts, autografts, and synthetic biomaterials, but all approaches present challenges including donor-site morbidity, tissue rejection, graft failure, and transmission of infectious diseases.¹³⁸ As a result, alternative methods such as cell-based therapies with MSC or MSC-like cells are being explored as adjuncts to surgical approaches.

The majority of studies regarding tendon and ligament healing have used BMD-MSCs, AD-MSCs, endogenous ligament-derived stem cells, and tendon-derived stem cells (TD-SCs). These studies also investigate a variety of delivery approaches including direct injection with or without collagen gel or tissue-engineered scaffolds seeded with MSCs to provide immediate mechanical support to injuries.¹³⁵ Roth et al¹³⁹ recently described that the therapeutic effects of locally applied MSCs are influenced by the cellular and molecular microenvironment at the injury site, in a bidirectional fashion. Modulating macrophages and promoting M1/M2 macrophage phenotype switch with MSCs plays a crucial role in reducing inflammation in injured tendons and joints. Based on this, it is reasonable to pursue future investigations relating to priming regimes of MSCs and tailoring to specific cell types or ECM components. Animal models investigating application of MSCs for ligament healing have focused on the anterior cruciate ligament (ACL) and generally show promise. Kanaya et al¹⁴⁰ performed intra-articular injection of BMD-MSCs in Sprague-Dawley rats following surgical partial transection of the ACL. At 4 week follow-up, there was healing tissue within the transection gap compared to the contralateral side that received only ACL transection. Oe et al¹⁴¹ demonstrated similar positive outcomes when they demonstrated improved biomechanics and histology after administering MSCs in rats with partial ACL transection compared to saline control. In addition, the equine superficial digital flexor tendon and the human Achilles tendon have demonstrated numerous similarities in structure, function, and injury pathophysiology. Specifically, it has been shown that intralesional injection of MSCs in equine models has produced favorable effects, likely enabled by granulation tissue and the enclosed nature of core lesions that may have provided an appropriate scaffolding opportunity. It is hypothesized that a delivery vehicle may allow for various types of scaffolding or 3D tendon-like tissue constructs may improve stem cell retention at the site of injury.¹⁴² One RCT demonstrated that AD-MSCs treatment might lead to increased collagen cross-linking of remodeling scar tissue in the equine population.¹⁴³ Another smaller study also showed that AD-MSCs and PRP can be considered a safe and effective strategy for the treatment of superficial digital flexor tendonitis in horses.¹⁴⁴

In comparison to the number of animal studies, there is a relative lack of translational human studies involving MSCs and ligament repair. One investigation performed by Wang et al¹⁴⁵ provides promising evidence evaluating the efficacy of MSC augmented ligament reconstruction. A double-blinded RCT examined 17 patients that received a single intra-articular injection of 75 × 10⁶ allogeneic immature mesenchymal precursor cells (MPCs) in a HA medium coupled with ACL reconstruction. Patients that had associated meniscal injury requiring one-third resection or reconstruction that would alter usual ACL rehabilitation were excluded. At 24-month follow-up, patients who received MSCs demonstrated significantly higher improvements in KOOS and pain scores. In addition, these patients were found to have reduced lateral tibiofemoral joint space narrowing at 24 months (Figure 4). The study did not, however, incorporate measurements of ACL healing such as clinical graft laxity grading or MRI signs demonstrating graft incorporation.¹⁴⁶

Another study was performed by Moon et al¹⁴⁷ examining the outcomes of UCB-MSCs in enhancing tendon-graft healing following ACL reconstruction (ACLR). The study enrolled 27 after the exclusion of 3 patients. In the experimental group, UCB-MSCs were suspended in a HA mixture and applied to tendon-bone interfaces of femoral tunnels during ACL reconstruction. A HA only group served as the control. There were no significant differences in the groups when measuring KT-2000 Knee Arthrometer (KT-2000) measurement, pivot shift, or International Knee Documentation Committee (IKDC) scores.

With regards to MSCs and tendon healing, the majority of studies have focused on rotator cuff repair. A study by Gulotta et al¹⁴⁸ used BMD-MSCs to augment supraspinatus repair compared to supraspinatus repair alone in a rat model. There was no difference in load-to-failure testing at 30 days but a significantly increased strength at the insertion site in animals receiving BMD-MSCs at 45 days. Ina separate study, Hernigou¹⁴⁹ demonstrated improved healing rates and a lower subsequent tear rate in a case-controlled study that involved applying 51,000 ± 25,000 autogenic BMD-MSCs during arthroscopic full-thickness supraspinatus repair.

Two clinical trials have been completed in the last 5 years employing MSCs in the treatment of tendinous injuries.^150,151 Another 5 clinical trials are either active, recruiting, or planning to recruit participants to investigate healing using MSCs (Table 4).^152–156 At this stage, while minimal adverse events have been reported, the literature is not convincing for the use of MSCs in the treatment of tendon or ligamentous injuries.^150,151

Applications of MSCs in treatment of intervertebral disc disease

Degenerative disc disease (DDD) is an umbrella term for intervertebral disc diseases, generally referring to pathologies of the lumbar spine from lumbar disc herniation or other discogenic low back pain. This collection of common diseases that increase with age and are growing in prevalence, has also been a target for treatment with MSCs.¹⁵⁷ A 2021 meta-analysis of RCTs demonstrated that MSC therapy could decrease VAS scores and Oswestry Disability Index scores, suggesting potential for MSCs as a novel therapeutic intervention for DDD.¹⁵⁸ The pathological processes of DDD are complex and multifactorial, but are comprised of immunological components involving various IL and tumor necrosis factor-alpha (TNF-α), both of which are implicated in the repair of discogenic disease by stem cells cocultured in an IDD-like environment.¹⁵⁹

Specifically, at least 5 types of cells have been identified as potential treatment vectors for IDD: BMD-MSCs, AD-MSCs, UCB-MSCs, intervertebral-derived stem cells IDVSCs, and pluripotent stem cells (PSCs).¹⁵⁷ BMSCs possess the capability to repair degenerated IVD tissues through the promotion of cell proliferation and enhancement of ECM components, including COL I and proteoglycans, while exosomes from BMD-MSCs can increase ECM production and encourage the expression of NP cell-related genes, ultimately achieving the desired effect of IVD self-repair.¹⁶⁰ AD-MSCs, like BMD-MSCs, have the capacity to differentiate into NP cells, generate distinct growth factors, and contribute to IVD restoration at various levels.²⁵ Several studies indicate that AD-MSCs are better suited than BMD-MSCs for the treatment of DDDs due to their superior differentiation potential, but they may also exhibit inferior endochondral osteogenic ability when compared to BMD-MSCs.^161–163 Like other MSCs, UCB-MSCs are relatively new and have strong proliferation and differentiation abilities, low immunogenicity, and no tumorigenicity.^164–166 Beeravolu et al¹⁶⁷ demonstrated that differentiation of UCB-MSCs into chondrogenic progenitor cells in vitro resulted in significant improvements in cell structure, ECM, and glycosaminoglycan content compared to original UCB-MSCs. Furthermore, other studies have indicated that UCB-MSCs promote ECM formation by significantly increasing proteoglycan and COL II expression in the NP.¹⁶⁸

The applications of IVDSCs face challenges, including low efficiency in separating cartilage endplate stem cells (CESCs) from cartilage endplates (CEPs), difficulty inducing CESCs to differentiate into hyaline cartilage tissue, and a harsh microenvironment of the IVD that poses a challenge to the viability of implanted stem cells.¹⁵⁷ PSCs are stem cells with high self-renewal, proliferation, and differentiation abilities, and include IPSCs and ESCs. IPSCs are generated by inducing the expression of recombinant transcription factors, while ESCs are derived from early embryos and can differentiate into all 3 germ layers.^169,170 However carcinogenicity, along with legal and ethical issues have limited the use of PSCs thus far. Presently, the greatest promise for stem cell therapy seems to be with BMD-SCs, AD-MSCs, and UCB-MSCs, with the question of optimal dose, frequency, time, and route of MSC transplantation at different stages of DDD needing to be explored in various clinical trials.¹⁵⁸

In the last 10 years, there have been 3 completed clinical trials and 3 additional clinical trials currently registered to investigate the treatment of IDD with MSCs. One of which reported results showing the use of BMD-MSCs directly injected into the nucleus pulposus decreased lumbar pain VAS scores at all time points, decreased sciatic pain VAS scores at 6 and 12 months, and increased water content on MRI in lumbar discs at 12 months.¹⁷¹ Two other studies are exploring the use of various umbilical MSC types, which could theoretically be superior in restoring disc height in comparison to other cell types due to their actions on the ECM and prior benefits in cell structure.^{167,172–176} Lastly, there is one trial assessing safety of MPCs, which have not been explored as much as other cell types and may provide a new avenue for the stem cell treatment of IDD (Table 5).¹⁷⁴

Conclusion

The application of cell-based therapies with MSCs in musculoskeletal diseases is a burgeoning and promising area of medicine. MSCs have the potential to provide a diverse and versatile means of treating musculoskeletal diseases. Multiple animal and human models have demonstrated promising results in treating orthopedic injuries/diseases driven largely by the promotion of regeneration of tissues; however, studies to-date have not demonstrated consistent efficacy and further research is required to confirm their safe clinical applications. MSCs can be extracted from a variety of tissues, and biochemical experiments are unraveling the benefits and possible specific roles of specific tissue harvestings. One of the most attractive properties of MSCs is their anti-inflammatory and immunomodulatory effects. However, there are ongoing obstacles to their usage. The majority of studies to-date lack standardization of cell extraction, expansion, preparation, and delivery, which makes a comparison of studies and homogeneous results difficult. In addition, there is ongoing controversy and concern regarding MSCs’ potential risks of tumor formation; however, human trials using MSCs have established their safety in trials so far. Another issue is the relative paucity of human trials, which requires further research to evaluate the safety, efficacy, and applicability of MSCs in orthopedic surgery and the treatment of musculoskeletal diseases. Despite these challenges, progress is being made, and the potential for MSCs to enter the therapeutic armamentarium for musculoskeletal disease remains promising. The application of MSCs has the potential to revolutionize the way we approach and manage musculoskeletal diseases.

Author contributions

Justin Trapana: Conception and design, Administrative support, Collection and/or assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. Jonathan Weinerman: Conception and design, Administrative support, Manuscript writing, Final approval of manuscript. Danny Lee, Anil Sedani, David Constantinescu: Conception and design, Administrative support, Final approval of manuscript. Thomas M. Best, Francis J. Hornicek, Jr.: Conception and design, Administrative support, Data analysis and interpretation, Final approval of manuscript. Joshua M. Hare, MD: Conception and design, Administrative support, Data analysis and interpretation, Manuscript writing, Final approval of manuscript

Conflicts of interest

J.M.H. reports having a patent for cardiac cell-based therapy and holds equity in Vestion Inc. and maintains a professional relationship with Vestion Inc. as a consultant and member of the Board of Directors and Scientific Advisory Board. Vestion Inc. did not play a role in the design, conduct, or funding of the study. J.M.H. is the Chief Scientific Officer, a compensated consultant and board member for Longeveron Inc. and holds equity in Longeveron. J.M.H. is also the co-inventor of intellectual property licensed to Longeveron. Longeveron did not play a role in the design, conduct, or funding of the study. The University of Miami is an equity owner in Longeveron Inc., which has licensed intellectual property from the University of Miami.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.