**2. MSCs in nonunion bone fracture**

Inadequate healing can lead to nonunion of the fractured bone. Clinically, about 5–10% of all fractures end up in persistent nonunion [10]; therefore, nonunion is one of the most troublesome complications. Since MSCs have the osteogenic differentiation ability, can secrete a variety of cytokines and promote angiogenesis. It is reasonable to speculate that MSCs could accelerate fracture healing. In fact, there are experimental evidences to support the idea that MSCs treatment indeed promoted healing of nonunion fractures. For example, MSCs has been transplanted to animals to promote bone loss and fracture healing [11, 12]. Also, there are reports of BMSC treatment of bone nonunion caused by a bone defect, osteogenesis and local microenvironment disorders in patients [13–15]. It was reported that, after traumatic injuries, BMSCs could migrate from blood circulation to the lesion site, and then directly differentiate locally, and replace the injured cells. Consistently, the circulating BMSC can be detected in peripheral blood 39 to 101 hrs after fracture [16]. However, other data also suggested that these circulating cells account for only a small portion of cells in the fracture callus under normal circumstances, suggesting that the majority of the cells at the fracture site are migrated from the adjacent tissues [13]; nevertheless, therapeutic amplification of circulating MSCs through their mobilization could also represent a potential therapeutic opportunity in fracture repair [13].

similar populations, including mesenchymal stromal cells (MSCs), mesenchymal progenitor cells, multipotent mesenchymal stromal cells, bone marrow stromal cells (BMSCs), bone marrow-derived MSCs, multipotent stromal cells, mesenchymal precursor cells, and skeletal stem cells [3]. Currently, most investigators prefer an alternative term, that is, multipotent mesenchymal stromal cells (MSCs). For this reason, we also use this term throughout this chapter. Theoretically, self-renewal without significant loss of their characteristics (stemness) and multi-lineage differentiation potential are the two criteria that define MSCs as real stem cells, but in practice, this heterogeneous population proliferates in vitro as plastic-adherent cells, as fibroblast-like morphology, forms colonies in vitro and can at least differentiate into bone, cartilage and fat cells [4]. In addition, literatures also provided evidence that MSCs can differentiate into multiple other mesenchymal lineages or even non-mesenchymal cell types, including endothelial cells, osteoblasts, chondrocytes, fibroblasts, tenocytes, vascular smooth muscle cells, myoblasts, and neurons [5], though some of these capacities are controversial. The key caveat is that it is unlikely that all cells in the cultural meet the above-mentioned two criteria. MSC-like cells can be obtained from almost all tissues, including the umbilical cord, amniotic fluid, placenta, adipose tissue, joint synovium, synovial fluid, dental pulp, endosteum, and periosteum [6]. Cultured MSCs have been characterized either by using cell surface antigens and/or by examining the cells' differentiation potential. The International Society for Cellular Therapy recommended that cells should fulfill the following criteria to be considered as MSCs: (1) the cells must be plastic adherent when maintained under standard culture conditions; (2) they must express CD73, CD90, and CD105 markers and should not express CD34, CD45, CD14, HLA-DR, CD11b, or CD19; and (3) they should be able to differentiate at least into osteoblasts, chondroblasts, and adipocytes in vitro [7]; however, this criteria has obvious problems therefore not been commonly accepted. For this reason, it is still challenging to consistently isolate or purify a well-defined clinical applicable MSC population.

210 Stromal Cells - Structure, Function, and Therapeutic Implications

On the other hand, the increasingly aging population has made the degenerative, non-traumatic and traumatic musculoskeletal diseases main socioeconomic issues, and MSCs seem to be a promising solution. In fact, MSCs have been widely used as a treatment for numerous orthopedic diseases, including bone defects, osteoarthritis (OA), femoral head necrosis, degenerative disc, spinal cord injury, knee varus, osteogenesis imperfecta, and other systemic bone diseases [8]. Currently, orthopedic researchers are still focusing on overcoming a variety of challenges so that they can fully realize the clinical therapeutic potential of MSCs, and the long-term goal is to change the main treatment strategy in the field of orthopedics from surgi-

In this chapter, we will briefly update the main advancements in these areas and discuss the

Inadequate healing can lead to nonunion of the fractured bone. Clinically, about 5–10% of all fractures end up in persistent nonunion [10]; therefore, nonunion is one of the most troublesome complications. Since MSCs have the osteogenic differentiation ability, can secrete a

cal replacement and reconstruction to bioregeneration and prevention [9].

major current and potential future applications pathways.

**2. MSCs in nonunion bone fracture**

Indirectly, BMSCs promote bone healing mainly through the secretion of bioactive molecules and extracellular membrane vesicles, which induce angiogenesis, regulate inflammation, inhibit apoptosis, and regulate osteogenesis differentiation. Since defective blood supply (ischemia) is an important cause of nonunion of bone, promoting blood vessel formation is beneficial to the healing of the nonunion bone. MSCs are known to secrete angiogenesisrelated factors include angiopoietin Ang-1 and Ang-2, vascular endothelial growth factor (VEGF), FGF-2, and hepatocyte growth factor (HGF)-1 [17]. Furthermore, BMSCs have an anti-fibrotic effect and can limit that fibrosis progression of fracture zone and promote the regeneration of bone tissue. This is mainly accomplished by immunoregulating, inhibiting TGF-3 mediated differentiation of fibroblasts, inhibiting oxidative stress, and matrix reconstruction [18]. Interestingly, it was also found that HGF, VEGF, and microbubble secreted by BMSCs had an anti-apoptotic effect, which inhibits the apoptosis of transplant cells in the injured area [19, 20].

In addition, other conserved signaling pathways, such as the transformation growth factor (TGF)-β<sup>3</sup> /bone morphogenetic proteins (BMP), Wnt, Hedgehogs, FGF, platelet-derived factor (PDGF), epidermal cell growth factor (EGF), and insulin-like growth factor (IGF), may also indirectly participate in the regulation of BMSCs and promote bone healing processes [21, 22]. Based on these observations, factors such as TGF-β<sup>3</sup> and its analogs, BMP, BMP-2, and BMP-7, have been used clinically to enhance and accelerate the bone repair or regeneration.

Technically, MSCs can be isolated from many different tissues. The iliac crest is the ideal position for bone marrow aspiration. In clinical practice, we indeed found that injection of bone marrow aspirate into the fracture space can promote the healing of fracture and shorten the healing time. For example, in one case with an open tibial fracture, which did not develop callus within the 6 months after surgery, and then we extracted marrow aspirate from the iliac crest and injected it into the fracture space. We found that the fracture healed well 6 months after the transplantation (**Figure 1**). Consistently, bone marrow aspirate injection has been shown to have a potential role in the treatment of aseptic, atrophic nonunions with acceptable alignment and minimal gap, or displacement between fracture fragments [15]. Generally, Tibial nonunion treatment with bone marrow aspirate has been well-documented and found to be successful in 75–90% of reported tibial nonunion case series [23, 24].

**Figure 1.** (A) A 32-year-old male underwent external fixation of an open tibial fracture. After surgery, no callus was observed in 6 months. (B, C) he underwent bone marrow aspiration and percutaneous grafting directly into the nonunion site. (D, E) 6 months after the transplantation, frontal and lateral X-ray images indicated the fracture healed well.

Researchers have described the purification and expansion of bone marrow MSCs from mice, rats, rabbits, dogs, and humans, and the ability of these cell populations to form bone when implanted ectopically with hydroxyapatite or an appropriate carrier has been established. To isolate MSCs from blood, mobilization of MSCs to the peripheral circulation with granu

Clinical Applications of Mesenchymal Stromal Cells (MSCs) in Orthopedic Diseases

http://dx.doi.org/10.5772/intechopen.76868

locyte colony-stimulating factor (G-CSF) is normally necessary. Data also suggests that the tissue-engineered constructs with MSCs (either genetic modified or not) brings us closer to a clinical application. However, nonunion still occurred in nearly half of the bone defects in a large animal model [25]. In fact, the commonly accepted idea that atrophic nonunion is due to lack of MSCs activities of MSCs might be not accurate. For example, an interesting study found the existence of MSCs (confirmed by their expression profile of CD105, CD73, HLA-DR, CD34, CD45, CD14, and CD19) in the site of atrophic nonunion, at a similar num

ber and viability to those isolated from the iliac crest [26]. In another clinical study, Ismail et al. [14] also reported that iliac crest autograft with or without autologous MSCs (with 5 g/

Due to the limited ability of proliferation capacity of chondrocytes, articular cartilage injury often causes progressive degeneration of the joint and OA, which is a serious health and economic problem [27]. The typical current treatment for this disorder is microfracture, which is a surgical technique that was developed 20 years ago. This treat

ment uses the body's own healing abilities to regenerate the chondral surface. However, the regenerated fibrocartilage often has poor mechanical properties compared with nor

Recently, the MSC-based autogenous transplantation treatment was proposed, since the potential of the MSCs to differentiate into chondrocytes has been well-recognized [28]. Compared with allogeneic cells, generally, autogenous cartilage progenitor cells are more effective in the treatment of articular cartilage defect [29]. The emerging typical paradigm to apply MSCs in this disorder is [30]: (1) during the first operation, a cartilage biopsy is taken from areas of damaged cartilage within the ankle or knee; (2) chondrocytes are isolated from the biopsy via enzymatic digestion and cultured in 2D monolayer cultures; (3) monolayer culture-expanded chondrocytes are seeded on a collagen type I–III membrane; and (4) in the second operation, the cartilage lesion is prepared and the collagen membrane is cut to size,

To clarify whether donor MSCs indeed contribute to cartilage regeneration in vivo via a progenitor-mediated mechanism [31], Zwolanek et al. describe a novel cell tracking system based on genetic transgenic donor and corresponding cell marker, and the results showed that MSC could contribute to cartilage regeneration via a progenitor - or nonprogenitor mediated mechanism [31]. The study by Windt et al. in humans also produced similar results [32]. Further study found that chondrogenesis can be regulated by adjusting the time and

hydroxyapatite granules, as scaffold carrier) had similar treatment efforts on atrophic

cm 3

nonunion.

mal cartilage.

**3. MSCs in articular cartilage injury**

placed in the lesion and secured with fibrin glue.

concentration of TGF-β [33].


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Researchers have described the purification and expansion of bone marrow MSCs from mice, rats, rabbits, dogs, and humans, and the ability of these cell populations to form bone when implanted ectopically with hydroxyapatite or an appropriate carrier has been established. To isolate MSCs from blood, mobilization of MSCs to the peripheral circulation with granulocyte colony-stimulating factor (G-CSF) is normally necessary. Data also suggests that the tissue-engineered constructs with MSCs (either genetic modified or not) brings us closer to a clinical application. However, nonunion still occurred in nearly half of the bone defects in a large animal model [25]. In fact, the commonly accepted idea that atrophic nonunion is due to lack of MSCs activities of MSCs might be not accurate. For example, an interesting study found the existence of MSCs (confirmed by their expression profile of CD105, CD73, HLA-DR, CD34, CD45, CD14, and CD19) in the site of atrophic nonunion, at a similar number and viability to those isolated from the iliac crest [26]. In another clinical study, Ismail et al. [14] also reported that iliac crest autograft with or without autologous MSCs (with 5 g/ cm3 hydroxyapatite granules, as scaffold carrier) had similar treatment efforts on atrophic nonunion.
