Recent advances toward understanding the role of transplanted stem cells in tissue-engineered regeneration of musculoskeletal tissues

Bone tissue engineering

The current gold standard of treatment of the critical-sized bone defect is the cancellous autograft, often harvested from the iliac crest¹. Autografts repair bone via osseointegration and osteoconduction, but the use of these grafts is limited by donor site morbidity, risk of infection, risk of surgical complications, and limited available bone volume²; thus, tissue engineering provides a promising alternative. Most musculoskeletal tissue engineering studies operate on the premise that stem cells seeded into biomaterial scaffolds and implanted into volumetric defects survive the ensuing ischemic microenvironment, differentiate into osteoblasts or myocytes, and integrate with the native matrix to directly impact tissue regeneration³. Hence, technologies such as in situ bioprinting are continually being advanced to precisely control the spatial location of cells and assess the impact of different “geometries”⁴. Other recent studies to achieve bone healing examined the effect of stimulating endochondral ossification with ASCs differentiated into hypertrophic chondrocytes and implanted into a rat femoral defect. They hypothesize that implanted hypertrophic chondrocytes help to both regulate endogenous cell behavior and directly contribute to bone formation; however, they did not determine whether improved bone formation was predominantly due to the survival and integration of the implanted cells or to their superior secretory and immunomodulatory properties⁵. A study by Larson et al. demonstrated that contact with viable bone shifted the phenotype of chondrogenically pre-cultured MSCs to hypertrophic and osteogenic phenotypes in three-dimensional (3D) cultures of MSCs and in a nude rat model⁶. Implanted chondrogenically pre-cultured MSCs, but not non-differentiated MSCs, seeded on polycaprolactone (PCL) scaffolds exhibited mineralization, formation of trabecula-like structures, and chondrogenic and osteogenic gene expression profiles at 8 weeks. Interestingly, scaffolds seeded with chondrogenically pre-cultured MSCs were the only group with human DNA present at 8 weeks and with 45% human RNA content. The findings from these studies suggest that the direct contribution of transplanted cells to bone regeneration might depend heavily on the pre-implantation priming^5,6.

Skeletal muscle tissue engineering

A variety of stem cell types have been explored as a potential source of myogenic cell replacement therapy. ASCs, which provide a high rate of stem cell proliferation and can potentially be sourced directly from the patient, bypassing immune rejection, have been evaluated extensively in the treatment of musculoskeletal damage^7–12. However, recent studies have called into question whether ASC-derived cells are contributing to de novo myofiber regeneration directly: Gorecka et al. injected autologous ASCs into the tibialis anterior (TA) of mice following a crush injury and found that while the cell transplantation resulted in increased fiber cross-sectional area and improved muscle contractility, ASC-derived cells did not differentiate into myofibers or fuse with endogenous muscle fibers¹³. Similarly, Gilbert-Honick et al. seeded ASCs onto electrospun fibrin hydrogels and transplanted this construct into a mouse model of VML injury¹⁴. Although there was an increase in fiber cross-sectional area, only limited expression of myogenic markers in the donor cells was observed. Alternative mechanisms for the therapeutic benefit of ASC transplantation have been posited. Secretion of paracrine factors by transplanted stem cells may improve regeneration by activating endogenous muscle stem cells, by reducing inflammation at the site of the injury, or by promoting angiogenesis^15–17. In particular, a number of recent studies have documented anti-fibrotic effects of transplanted stem cells. Di Summa et al. found that differentiated ASCs incorporated into fibrin nerve conduits and transplanted into a rat nerve gap model reduced fibrotic tissue formation, enhancing axonal regeneration and remyelination¹⁸. Milosavljevic et al. injected MSCs or MSC-conditioned medium intravenously into mice and demonstrated that CCl4-induced liver fibrosis was attenuated¹⁹. They found that MSCs acted on fibrosis by means of decreasing levels of inflammatory T helper 17 (Th17) cells while increasing anti-inflammatory CD4⁺ interleukin 10–positive (IL-10⁺) T cells. These anti-fibrotic effects of ASCs and MSCs may play a role in their regenerative capabilities upon transplantation into injury models.

A number of recent studies have explored the use of human iPSCs as a source of myogenic cell transplantations. Multiple groups have demonstrated that iPSC-derived myogenic cells contribute directly to the formation of new myofibers in damaged tissue^20–22. Rao et al. generated the first 3D contractile skeletal muscle constructs from human iPSCs²³. After transplantation, the cells formed densely packed, aligned myofibers and retained functional responses. Wu et al. injected iPSC-derived myogenic progenitors into cardiotoxin-injured mouse TA and observed engraftment and contribution of the iPSC-derived cells to new myofiber formation²⁴. However, long-term survival of iPSC-derived cells in the transplanted environment is a limitation, multiple groups have reported high levels of cell death upon transplantation^21,23, and further work is necessary to characterize the therapeutic mechanism of transplanted iPSC-derived cells. An emergent alternative to the treatment of VML is the use of autologous minced muscle grafts, or 1 mm³ pieces of muscle, which have also been demonstrated to attenuate T lymphocyte and macrophage responses to severe muscle injury. These results may indicate a promising therapeutic role for cell aggregates and immunomodulatory therapies in the treatment of VML²⁵.

Stem cell aggregates

Implanting stem cells as aggregates rather than monodispersed cells has been shown to enhance their viability, migration, and differentiation and modifies their secretion of cytokines, immunomodulatory factors, and EVs to improve therapeutic outcomes^29,30. Yet the lack of standardization renders it impossible to accurately correlate the impact of aggregate sizes, methods of aggregation, and mode of implantation on the therapeutic outcomes. Recent investigations into bone tissue engineering have used periosteum-derived stem cells embedded in collagen type 1 hydrogel³¹, MSCs embedded in a platelet-rich plasma construct³², or arginine-glycine-aspartic acid (RGD) functionalized alginate³³. The studies employed 250 cells per aggregate, randomly sized spontaneous aggregates, and 500 cells per aggregate, respectively. Aggregation increased osteogenic and chondrogenic markers and paracrine secretions. When compared with monodispersed cell-laden counterparts, aggregate-laden scaffolds showed increased bone formation³² but no beneficial impact on cell survival or construct vascularization when implanted subcutaneously³¹. However, when stem cell aggregates were used in conjunction with bone morphogenetic protein 2 (BMP2) stimulation, there was increased blood vessel formation, BMP2 production, presence of hypertrophic chondrocytes, and remodeling³¹, although there was no increase in bone volume or torsional strength of the resulting bone³³. For skeletal muscle tissue engineering, recent studies have tested human umbilical cord–derived MSCs³⁴ as aggregate sheets encasing porcine heart decellularized extracellular matrix and green fluorescent protein (GFP)-labeled murine MSCs³⁵ (500 cells per aggregate) injected in phosphate-buffered saline, respectively. In both cases, the investigators observed increased recovery and peak isometric torque observed from aggregates compared with single cells³⁵.

Extracellular vesicles/exosomes

EVs such as exosomes or microvesicles carry important cargo for cell communication and are influential in cell signaling. Exosomes and microvesicles are distinguished by their sizes and origins (that is, endocytic pathway versus plasma membranes). They both contain lipids, nucleic acids, and protein cargo. Studies have shown that EVs can enhance cell differentiation and viability, which in turn may affect therapeutic outcomes, although the molecular underpinnings of this are not yet understood. It has recently been suggested that EVs are heavily involved in bone homeostasis³⁶ and skeletal muscle myogenesis³⁷, but further investigations are necessary in order to elucidate the mechanisms in which these occur. For bone tissue engineering, recent studies of MSC-derived EVs or exosomes embedded in a hydrogel for treating critical-sized calvarial defect have demonstrated therapeutic benefit with significant increases in bone volume fraction, bone mineral density, and new bone area^38–40. Furthermore, combining EVs with MSCs resulted in increased bone volume and bone volume fraction compared with either component delivered separately⁴¹. To regenerate skeletal muscle, recent studies have explored the use of ASC-derived EVs^17,42 and MSC-derived exosomes¹⁵ injected at various time points at the site of injury or intravenously. These studies also measured various outcomes from an increase in cross-sectional area of newly formed fibers¹⁷ to increased regulation/expression of myogenic genes⁴², capillary density, myofiber diameter, number of centrally located nuclei, and decreases in fibrotic area¹⁵.