Mesenchymal Stem Cells in Regenerative Medicine for Musculoskeletal Diseases: Bench,Bedside,and Industry

Basics of MSCs

Bone marrow, the home organ of hematopoietic stem cells, contains several subpopulations of stem/progenitor cells that are capable of differentiating into various non-hematopoietic cells. Among the best studied subpopulations are the cells referred to as marrow stromal cells, mesenchymal stem cells, or multipotent mesenchymal stromal cells (MSCs) (27). Such cells can be isolated by exploiting their ability to adhere to plastic tissue culture surfaces. MSCs have been identified from a variety of other tissues, including, but not restricted to, adipose tissues, Wharton's jelly of umbilical cord, and dental pulp (12). In addition, MSCs have emerged as a promising tool for clinical and commercial applications of cell transplantation and cell-based therapy, such as tissue engineering. In fact, the world's first stem cell drug uses MSCs to treat children suffering from graft-versus-host disease (GVHD) in allogeneic recipients (62). It has also been recently reported that most stem cell therapies on the market and in development utilize MSCs. Ultimately, there are numerous reasons why MSCs have attracted so much interest in clinical applications and industry, including ease of isolation from patients, expandability in culture with maintained differentiation potentials, immune-modulating properties, and limited tendencies to form tumors.

Criteria for Defining MSCs

The therapeutic potential of MSCs has created remarkably growing interest in a wide variety of biomedical disciplines but has also generated increasing difficulties in comparing outcomes. As a result, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) has proposed three criteria to define MSCs (17). First, MSCs must be plastic adherent when maintained in standard culture conditions. Second, MSCs must express cluster of differentiation 105 (CD105), CD73, and CD90 and lack expression of the markers CD34, CD45, CD14 or CD11b, CD79a or CD19, and human leukocyte antigen (HLA)-DR in culture.

Third, MSCs must have the potential to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro.

Differentiation Potentials of MSCs

MSCs were first demonstrated to potentially differentiate into osteogenic, adipogenic, and chondrogenic lineages in vitro (52) and in vivo (40). They also possess the ability for musculoskeletal and cardiomyocyte differentiation (40,64). In addition, MSCs have been shown to differentiate into not only mesenchymal but also nonmesenchymal lineages. For instance, MSCs have been differentiated into neuronal cells both in vitro (29) and in vivo (36). Furthermore, hepatocytic differentiation has also been observed in vitro (39) and in vivo (58). Additionally, there are many experimental animal models that utilize MSCs in cell transplantation or cell-based therapy in regenerative medicine of diseases, including musculoskeletal disorders (20), myocardial infarctions (44), hepatic failures (32), as well as neural degenerations such as spinal cord complications (76) and Parkinson's disease (14).

Paracrine Effects

Although the therapeutic effects of MSCs have been demonstrated in a variety of disease models and clinical trials, the engraftment and differentiation rates of MSCs into host tissues are extremely low. For example, MSCs engrafted in mouse bone to treat osteogenesis imperfecta resulted in an engraftment rate less than 1.5% (49). Moreover, the in vivo tracking analysis of cell transplantation was complicated by the cellular fusion mechanism (68,75) and background tissue-specific fluorescence (33). Thus, to explain the therapeutic effects of transplanted MSCs, which had low engraftment and differentiation rates, an MSC-induced paracrine effect was proposed—a major conceptual change to the role of MSCs in therapy (53). Instead of MSCs directly differentiating into the injured cells, it is suggested that MSCs secrete paracrine factors that elicit regenerative responses, a concept supported by using only conditioned medium or secretome to enhance angiogenesis (30), promote skin wound healing (79), stimulate fracture healing (74), repair nervous degeneration (63), and treat cardiovascular diseases (65). Furthermore, cumulative evidence shows that MSCs secrete growth factors and cytokines that regulate endogenous tissue regeneration as well as modulate the immune responses and inflammation of several diseases.

Preclinical and Clinical Studies of MSCs in the Treatment of Musculoskeletal Diseases

Musculoskeletal diseases (MSDs) are a group of disorders that result from trauma or degeneration in either a single event or repetitive episodes. With the potential to differentiate along mesenchymal lineages, MSCs have been widely used in cell-based therapy for the treatment of MSDs. There is a large amount of research findings on the use of MSCs for MSDs in experimental animal models and clinical settings, as reviewed previously (7,43,47,56,61). Most studies using MSCs for cell-based therapy in MSDs have shown promising results (18,25,26). We will discuss the current progress of MSC applications in scientific research and marketing availability regarding several MSDs.

Bone Regeneration

In preclinical studies, MSCs have been suggested to restore large bone defects in several animal models of disease (7,9). The effectiveness of expanded MSCs from different animals behaves in a similar manner for bone regeneration in vivo. Our group has also successfully applied rabbit MSCs to treat calvarial defects in allogeneic recipients, with the repair ability more obvious in MSCs expanded under hypoxic conditions than that expanded under normoxic conditions (78). Clinically, when treating osteonecrosis of the femoral head with autologous bone marrow aspiration, the quantity of mineralized bone formation correlated positively with the amount of MSCs residing in the iliac crest (24). Numerous studies have also investigated the use of ex vivo expanded MSCs in bone regeneration. For instance, three patients with segmental bone defects were successfully treated with autologous MSCs delivered in hydroxyapatite scaffolds (54). There are several investigations that integrated MSCs into biomaterials like hydroxyapatite and calcium phosphate and showed promising features, including their ease of availabilities, osteoconductivities, and absence of immune responses (6,57,69).

Cartilage Regeneration

Owing to cartilage's limited quantity, extracting autologous tissue from healthy cartilage is problematic. MSCs have been demonstrated to undergo chondrogenic differentiation when encapsulated in alginate both in vitro (42) and in vivo (41), suggesting the potential for cartilage regeneration. MSCs have also been shown to enhance cartilage repair in full-thickness defects of articular cartilage (55,71,77). A clinical study also reported that full-thickness articular cartilage defects in the patellofemoral joint transplanted with autologous MSC-embedded collagen gels showed significant and lasting restoration of cartilage after follow-ups at 17 to 27 months (73). When comparing autologous MSC transplantation with autologous chondrocyte implantation (ACI) for cartilage repair, the MSC transplantation was shown to be more economical, minimized donor-site morbidity, required less surgery, and still as effective as its ACI counterpart in a 2-year follow-up (45). When treating osteochondral lesions of talus (OLT), the clinical outcomes of MSC injections with arthroscopic marrow stimulation to OLT were superior to those with arthroscopic marrow stimulation alone, with the results measured using the visual analog scale (VAS) for pain and the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle–Hindfoot Scale for ankle activity (35).

Treatment of osteoarthritis (OA) is much more difficult, since the defect is larger in size and is characterized by an inflammation environment, so there is still a lack of long-term success in using MSCs to treat OA (61). MSCs embedded in collagen gels have been transplanted to treat patients with knee OA who only underwent a high tibial osteotomy, and the effects were compared to that of patients who underwent a high tibial osteotomy. Although the clinical improvement in each of these two groups was not significantly different, the cell-transplanted group achieved better arthroscopic and histological results than the cell-free control group (72). Another clinical trial involved patients of OA who received intra-articular injections of culture-expanded, bone marrow-derived MSCs, and 63.2% of patients showed improvement at an average follow-up of 11.3 months (10). However, these data still have to be considered preliminary, as another study using intra-articular injections of MSCs to treat knee OA reported only minor improvements in the ranges of motion (13).

Spinal Fusion and Disc Regeneration

While iliac crest autografting remains the gold standard in bone fusion studies, there are many disadvantages to autograft harvest including pain, bleeding, infection, and fracture risk. The preclinical animal models that use MSCs for spinal fusion provided the framework for clinical studies in spinal fusion (81). MSCs have been applied for spinal fusion as a component of Osteocel Plus, an alternative allograft cellular bone matrix, to treat patients who underwent a minimally invasive transforaminal lumbar interbody fusion for degenerative lumbar conditions. Twenty-one patients (91.3%) and 24 levels (92.3%) achieved radiographic evidence of solid bony arthrodesis at a 12-month follow-up (1). MSCs hybridized with β-tricalcium phosphate (β-TCP), another substitute for autografts, and utilized for lumbar spinal fusion resulted in a successful fusion rate of 93.3%, close to the fusion rate (96.2%) achieved by autologous iliac crest bone grafts (81). The regenerative ability of autologous MSCs has also been clinically investigated in degenerated intervertebral discs, where pieces of collagen sponge containing autologous MSCs were grafted. Symptoms were alleviated, and radiographic analysis showed improvements in the vacuum phenomenon and lumbar disc instability at a 2-year follow-up (80).

Tendon Healing

Although early clinical trials using MSCs in bone and cartilage repair are already published, information about MSC-based therapy in tendon repair is currently limited to animal studies. However, there are many promising preclinical studies that open up possibilities for future clinical trials. MSCs have been shown to enhance Achilles tendon repair in rabbit (11) and rat models (28,48). It is interesting to note that the healing effects of rat MSCs expanded under hypoxic conditions were superior to that of MSCs cultured under normoxic conditions (28). Unfortunately, MSC application in a rotator cuff repair model did not lead to improvement of structure, composition, or strength of the healing tendon attachment site despite evidence that MSCs were present and metabolically active (22). Nevertheless, there are also large-animal models that support the transition to human clinical trials. For instance, of the 113 racehorses that received intralesional autologous MSC injections for superficial digital flexor tendon strains, 111 (98.2%) returned to racing with a significantly lower reinjury rate than previous studies had obtained (21).

MSC-Related Products in Industry

It has recently been predicted that the stem cell therapy market was valued at US$2.7 billion in 2011 and is expected to reach an estimated value of $4.65 billion by 2016 (62). Noticeably, MSC-based cell therapy is still the major product type on the market and in development when compared to therapeutic techniques using embryonic stem cells, adult progenitor cells, and induced pluripotent stem cells. The market value of US osteobiologic products between 2009 and 2010 was about $1.6 billion, with the stem cell products accounting for $59 million. The sales of these products increased from 2009 to 2010 by 15.7%, making it a fast-growing subsegment in this market with an expected global value of $600 million by 2015. Thus far, the only three products that are MSC related and available in the US in this group are OsteoCel Plus, Trinity Evolution, and LiquidGen marketed by NuVasive Inc. (San Diego, CA, USA), Orthofix Inc. (Lewisville, TX, USA), and Skye Orthobiologics LLC (Redondo Beach, CA, USA), respectively. Meanwhile, two competitors are waiting for the green light to expand into the US market: Allostem and Cellentra Viable Cell Bone Matrix (VCBM), distributed by Allosource Inc. (Centennial, CO, USA) and Biomet Inc. (Warsaw, IN, USA), respectively.

OsteoCel Plus, an allograft cellular bone matrix containing MSCs and osteoprogenitor cells combined with demineralized bone matrix (DBM) and cancellous bone, is used in spinal fusion and other orthopedic surgical procedures. Owing to its osteoinductive, osteoconductive, and osteogenic capabilities, patients can be treated with the advanced bone graft product and avoid limitations of traditional alternatives. In 2010, OsteoCel Plus was the leading product in the osteobiologics market, accounting for over 92% of market sales. Business speculators believe that sales of OsteoCel Plus will continue to gradually increase, reaching $74 million in revenue by 2017 (70). Although OsteoCel Plus is the dominant product on the market, it is threatened by stem cell competitors like Trinity Evolution and non-stem cell opponents such as InFuse distributed by Meditronic Inc. (Sarasota, FL, USA). OsteoCel Plus has the current advantage due to its more advanced technology and first-to-market availability; however, its growth rate will be further hindered by increased competition against other upcoming stem cell-related products.

Trinity Evolution by Orthofix Inc., also a cell-based bone matrix used in spinal fusion surgery, supplies adult MSCs, osteoprogenitor cells, and a demineralized cortical component in a minimally manipulated bone allograft. The cancellous bone used to produce Trinity Evolution is derived from freshly recovered donor tissue and processed under aseptic conditions. Preclinical studies as well as strict donor screenings have demonstrated the safety of Trinity Evolution as well as its osteoinductive and osteogenic potential contained within a natural osteoconductive matrix. After a 5-year accumulation of over 75,000 cases with Trinity Evolution, Orthofix Inc. introduced Trinity ELITE in July 2013 as an even safer and fully moldable alternative that facilitates handling procedures in surgery.

Besides the use of viable MSCs in orthopedic products, there is ongoing development of supportive materials that each contains an MSC-secreted extracellular matrix and secretome. Skye LiquidGen is an allograft tissue matrix for use as an in vivo wound cover to fill tissue defects or localize areas of inflammation. One of its major components is the amniotic tissue-isolated collagen that acts as a natural scaffold for cellular attachment and accelerates cell migration and proliferation in vivo. LiquidGen can be applied directly to the surgical site, mixed with patients' own blood, or used with other carriers to cover or fill soft tissue defects. LiquidGen is also cryopreserved to extend its shelf life and is more convenient to handle in the operation room.

There are still several MSC-related products waiting to be released in the US market. The first products that emerged in the US were AlloStem from AlloSource Inc. and Cellentra VCBM from Biomet Inc. AlloStem Stem Cell Bone Growth Substitute, an adult human stem cell bone graft, is recovered from adult human adipose tissue. This minimally processed allograft is designed to promote bone growth and healing. Adipose tissue is another rich source of a variety of stem cells, and some laboratory studies even suggest that it is the human body's primary source of stem cells. Cellentra VCBM also offers a plethora of bone-healing factors comprised of an osteoconductive scaffold that harbors viable osteogenic cells and MSCs of verified osteoinductivities. It also provides additional inherent growth factors, including bone morphogenetic protein-2 (BMP-2), -4, -7, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), and fibroblast growth factor (FGF) to further improve bone growth. The cancellous bone matrix of Cellentra VCBM offers an interconnected trabecular structure for optimal osteoconductivity.

For markets outside of the US, the Korea Food and Drug Administration (KFDA) approved the manufacture and sale of Cartistem in the country for the treatment of traumatic and degenerative OA in January 2012. Cartistem contains specially selected and grown MSCs from the umbilical cord blood donated by willing mothers following KFDA regulation and guidelines. This makes Cartistem a safe stem cell-based allograft obtained from another individual rather than from the patient. However, Cartistem is currently only available in Korea through distribution by Dong-A Pharmaceuticals Co., Ltd. (Seoul, S. Korea) but has also received US Food and Drug Administration clearance to be tested in Phase I/IIa clinical trials for the possibility of US market expansion.

Current Problems and Perspective in Clinical Application of MSCs

There are several problems that should be resolved before MSCs are clinically tested (56). Cell transplantation of MSCs for clinical use requires a great number of cells, usually ranging from 10⁸ to 10⁹ depending on the disease treated. Currently, there is no gold standard for an MSC expansion process, as variations in expansion conditions induce different changes in cell properties (50). Expanded MSCs have also been reported to undergo proliferative senescence with loss of stem cell properties (4,8). More importantly, the degree of homing and engraftment of these expanded MSCs in adult recipients are very low (2,37,51). The paracrine effects of MSCs are also affected by aging after long-term expansion (34). Thus, there is great interest in the identification of methods that efficiently expand enough transplantable cells.

A good protocol that expands MSCs for clinical use should meet the following guidelines. First, MSC expansion should be in large scale and not suffering from senescence, loss of proliferation capacity, or loss of differentiation potential (4,8). Second, the expanded cells should engraft or regenerate tissues after transplantation into autologous or allogeneic recipients (17). Third, the therapeutic effects of engrafted MSCs are unknowingly due to engraftment and/or paracrine-induced effects, so this detail must first be confirmed before proceeding to clinical trials (53). There is controversy because, while MSCs have been shown to differentiate into injured cells, MSC-derived microvesicles have also been shown to protect against injuries like renal damage in mice (23). Finally, the final product must meet the ISCT minimum criteria for MSCs. In addition, the protocols and facilities used for cell manipulation and/or expansion should be regulated and approved to provide products that pass tests of identity, potency, purity, and safety (60,67).

Culturing MSCs at a low seeding density causes rapid expansion of MSCs (59); however, cells expanded at this seeding density reveal significant replicative senescence. Furthermore, ex vivo expanded MSCs failed to engraft, differentiate, or respond to host environments and also failed to secrete growth factors or cytokines (3). Recently, the beneficial effects of hypoxia on expansion of MSCs were found to be consistent even at different oxygen concentrations (19,46,66). Hypoxic cultures also enhanced the angiogenic effects of MSCs by increasing the secretion of angiogenic factors (30). Besides, in vitro migration and in vivo engraftment were increased by hypoxic exposure to MSCs for 1–2 days, detected by increases in chemokine (C-X3-C motif) receptor 1 (CX3CR1) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression (31). These results suggest that hypoxic culturing or short-term hypoxic preconditioning of MSCs may provide a general method of enhancing their survival, migration, angiogenesis, and engraftment in vivo in a variety of tissues.

The immunosuppressive features of human, baboon, and murine MSCs were demonstrated in vitro (5,15,16,38) and in vivo (5). MSCs have also been applied in allogeneic transplantation in diseases such as GVHD. Nevertheless, many studies have shown that allogeneic MSCs were rejected in immunocompetent major histocompatibility complex (MHC)-mismatched recipients, and thus, the use of MSCs for allogeneic transplantation remains controversial. However, a previous study demonstrated that allogeneic hypoxic MSCs increased the effects of bone defect repair compared to the effects produced by allogeneic normoxic MSCs (78). These results suggest that hypoxic MSCs are intrinsically immunoprivileged and can serve as a “universal donor cell” in treatments.

All together, the studies currently available suggest that expanded MSCs have multiple therapeutic effects on MSDs, which can be applied for bone regeneration, cartilage defect restoration, osteoarthritis treatment, spinal fusion, disc regeneration, and tendon repair. Based on these significant benefits, accumulating MSC-related osteobiologic products are available in the market or in development. However, there is still a lack of a gold standard procedure to expand MSCs. Recently, using a hypoxic culture has been shown to enhance short-term proliferation, long-term expansion efficiency, differentiation potential, stemness or maintenance of stem cell properties, expression of chemokine receptors, migration, and engraftment ability. Moreover, MSCs were able to survive and engraft in allogeneic recipients. The platform based on hypoxic culture will help the development of new strategies for clinical applications of MSCs.