• Stem Cells in Orthopedics

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     Application of Stem Cells in Orthopedics

    To differentiate between favorable application strategies the aim of treatment is one important factor. As mentioned before, MSCs have the potential to rebuild injured tissue but also to secrete growth factors for enhancing tissue regeneration. Depending on the underlying pathology, the treatment strategies differ considerably. In one patient a large tissue defect has to be filled by means of tissue engineering, whereas in another the substantial defect is bridged with residual tissue of low quality and only an improvement of healing environment is indicated.

    Besides the direct injection in the surrounding tissue, biomaterials are frequently used as carriers for drugs, bioactive molecules and cells. These materials have to fulfill some fundamental requirements. At first they have to be immune-compatible and nontoxic, whereas the degradation process must neither release toxic substances nor tissue-toxic concentrations of degradation products. For a later replacement with regenerated tissue, bio-degradable materials are important. The degradation velocity must be balanced as too fast and too slow are both detrimental. Beside these qualities, matrices formed from biomaterials must have distinct properties with regard to the desired kind of tissue. The prerequisite of mechanical strength, bioactivity, and kinetics of degradation and drug/cell release significantly varies between different repair tissues. Besides the used biomaterials themselves, the 3-dimensional structures of scaffolds have great influence on cell growth and differentiation. Scaffolds must be highly porous with interconnected pores of a diameter of at least 100 μm to allow ingrowth of cells and vessels [85]. Pore sizes between 100 and 400 μm are ideal.

    Despite the tissue engineering of bone, for which various inorganic materials, such as hydroxyapatite, calcium phosphate, calcium carbonate, or glasses was tested, mainly organic biomaterials have been investigated for scaffold formation. These are either naturally derived, for example, collagen, fibrin, agarose, alginate, gelatin, silk or hyaluronic acid, or produced synthetically. Synthetically produced organic biomaterials are mainly polyhydroxyacids such as polyglycolides or polylactides. To control kinetics of degradation, recent studies were performed employing hydroxyl acid copolymers. Thus, it has been tried to adapt kinetics of degradation to those of tissue regeneration.

    As these synthetic polymers often lack bioactivity, their surface was modified to alter cell adhesion, migration, differentiation, and proliferation in recent studies. Thus, they were coated or copolymerized with bioactive materials or functional groups were attached to the polymer chain before scaffold fabrication [86–88]. Apart from surface modifications with bioactive materials, scaffolds were coated directly with cytokines to control proliferation and differentiation of seeded cells [89]. Other authors describe the coating of scaffolds with genetic vectors to perform transfection of cells with different growth factors [90]. Biomaterials for tissue engineering can also carry drugs that prevent microbial colonization or control ingrowth of scaffolds into the surrounding tissue [91, 92].

    Tendon Repair

    Considering physiological properties of tendon tissue, an application technique via scaffolds with native extracellular matrix and the capability of cell seeding and adhesion would be ideal [93]. Based on this hypothesis, most of the current studies used scaffold application techniques. The few studies which favored direct application techniques injected the suspension of MSC into bone tunnels or on the bone surface before tendon refixation to improve tendon-to-bone healing [94, 95].

    Scaffold application techniques for tendons can be divided into gel suspensions, 3D scaffolds of solid tissue, and hybrid techniques. Gel suspensions offer a perfect 3D filling of the defect, but the reduced stability in comparison to stable matrices may result in loss of gel at the repair site due to erosion. In a rabbit Achilles tendon model, Chong et al. [96] used a mixture of fibrin sealant and bone marrow-derived mesenchymal stem cells. The fibrin sealant was injected into the tendon and the repair site was additionally covered with the agent. Fibrin incorporates the advantages of a clinical use over years including FDA approval, bone marrow-derived mesenchymal stem cells remain viable in fibrin and published data indicate that fibrin itself has no effect on tendon healing [97]. In this study no differences between fibrin and fibrin with MSC could be shown histologically. In the early healing phase (3 weeks), significantly improved biomechanical properties were documented but not in subsequent time periods (6 and 12 weeks). In a rat rotator cuff model, Gulotta et al. [98] also used MSC in a fibrin sealant and placed it between tendon and bone before refixation of the tendon. In this acute tendon repair model they did not find any significant histological or biomechanical differences after 2 or 4 weeks, respectively. Noteworthy, the same group recently succeeded in enhancing tendon healing in the same rotator cuff model, applying transfected MSCs using the embryonic transcription factor MT1-MMP and the tendon transcription factor scleraxis [99, 100]. With a collagen gel, Awad et al. [101] presented a further gel-based application technique. They fixed a collagen gel with different concentrations of MSC to suture material and filled a defect in the rabbits’ patellar tendon. After 12 and 26 weeks, significantly higher maximum stresses and moduli were documented compared to natural repair tissues. However, an adverse event was observed as there had been an increased number of intratendinous ossifications (28%). In comparison to the intact tendon only 25% of the ultimate load was reached with MSC. Regarding all groups, cell concentration had no significant influence on the outcome. This study group improved its application technique and presented a hybrid technique (MSC in a gel-collagen sponge composite) [102]. In the rabbit patellar tendon model, the biomechanical properties and cellular alignment were significantly improved in the MSC group after 12 weeks. A different matrix is presented by Omae et al. in in vitro and in vivo studies [103, 104]. Xenotendon slices with a thickness of 50 μm were decellularized and seeded with bone marrow stromal cells. The first results of the bundled construct in a patellar tendon rat model showed a survival of the stromal cells in all layers. In vivo results with MSC have not been published yet but the approach is promising.

    In conclusion, the application of MSC in tendon repair shows promising but inhomogenous results in animal models. Current in vivo data favor the culture of MSC into a tissue-engineered construct, with the advantage of primary stability and allowing the cells to produce their own extracellular matrix. But there is no consensus about the ideal carrier construct. Clinical data are not yet available for MSC application in tendon repair.

    Cartilage

    Besides autograft transplantation and autologous chondrocyte transplantation, current therapeutic concepts of cartilage defects include the recruitment of MSC. Drilling, abrasion, or microfracturing of the subchondral bone aims at the recruitment of MSC from the subchondral bone to stimulate the formation of cartilage repair tissue. In experimental and clinical studies of these standard techniques, a nonhyaline cartilage with high proportions of fibrous elements and inferior functionality has been found [105].

    For autologous cartilage repair various two- and three-dimensional constructs are available. Most of the matrices consist of natural polysaccharides and proteins, such as alginate and collagen. Furthermore, synthetic polymers are also available for example, polyethylene glycol (PEG) or polylactic acid (PLA). Successful outcome of a stem cell-based cartilage tissue engineering also depends on the design of extracellular matrix for a proper differentiation of MSCs into chondrocytes [106]. The most important property, namely, mechanical stability, to provide appropriate cell-matrix interactions to stimulate tissue growth and capability of functional tissue growth. The ideal matrix has sufficient strength to protect the cells from axial load and shear forces, is highly adhesive to remain stable in the repair site and possesses enough porosity to allow nutrient and differentiation factors to diffuse through it. Currently, a large number of in vitro studies focus on the optimal three-dimensional matrix.

    Increasingly innovative matrices are tested in in vivo animal models. For example, Shafiee et al. [107] performed MSC-based cartilage repair in a rabbit model with full-thickness cartilage defects. They used poly(vinyl alcohol)/polycaprolactone (PVA/PCL) nanofibers as matrix which showed a support of MSC proliferation and chondrogenic differentiation in vitro. The animals treated with MSC showed an improved healing of the defects compared with the untreated control. Tay et al. [108] used alginate-embedded MSC for the repair of focal cartilage defects in a rabbit model. They compared the macroscopic and histological results of MSC versus autologous chondrocyte transplantation 6 months postoperatively. MSCs had a similar effectiveness as chondrocyte transplantation, MSC even showed a significantly better macroscopic score. Both treatments resulted in superior tissue regeneration compared with untreated control defects. These promising results from the laboratory resulted in the first clinical studies about cartilage repair with support of MSC. The earliest data are case series of Wakitani et al. [109, 110]. They performed a bone marrow aspiration from the iliac crest and the MSC were expanded in culture. Four weeks later, the MSC were implanted using a collagen gel and the defect was additionally covered with a periosteal flap. The authors describe satisfying clinical and macroscopic results, but the small number of patients, the retrospective study design and the missing control has to be taken into consideration. Nejadnik et al. [111] performed a matched pair analysis of 36 patients in each group who underwent autologous cartilage transplantation or implantation of MSC. The postoperative followup after 24 months showed no significant difference of different functional knee scores between the groups.

    In the treatment of osteochondral lesions, the group of Buda et al. [66, 112] published clinical results of lesions in the femur condyle and the talus. In the talus group, MSC were taken from the iliac crest and incubated with a hyaluronic acid membrane () or collagen powder () before implantation in the defect in a single step procedure. 48 patients were examined clinically and radiologically after an average of 29 months postoperatively. The clinical scores revealed a significant improvement compared to postoperative scores whereas in the MRI and histology of second-look arthroscopies none showed complete hyaline cartilage. In the 20 patients with MSC therapy of the femur condyle satisfactory clinical results (IKDC 90.4 points) were also reported after an average of 29 months postoperatively. The MRI showed a satisfactory integration of the graft in 80% of the patients. Instead of direct defect coverage, some groups describe a simple intra-articular injection of MSC [113], with the intention of the ability of homing of the MSC. Centeno et al. report about an injection in a patient with early osteoarthritis of the knee. In the MRI followup after 6 months, they revealed an increased cartilage volume compared to point of time before injection.

    In summary, all applications for clinical use are based on very small case series. The MSC application technique was adopted from the clinical experience of autologous chondrocyte transplantation (fibrin, collagen gel, periosteal flap). Before a clinical use can be recommended, basic research to optimize application techniques, cell preparation, and concentration are essential [114]. With improved knowledge from basic studies further evaluation of the clinical potential of MSC application has to be performed in larger randomized controlled trials.

    Bone

    In bone, the main focus of regenerative medicine approaches lies on atrophic non union and replacement of lost bone tissue. Large bone defects are usually caused by trauma, infection, or tumors, as atrophic nonunion are usually caused by insufficient blood supply, interposition of soft tissue or consequence after infection. Current treatment strategies include autologous bone grafts from the iliac crest, which is actually the gold standard—and as salvage procedures—autologous fibula graft transfer and allogenic bone graft transplantation. However, all mentioned techniques show limitations, as bone supply is limited, autologous bone harvesting is accompanied with high rates of morbidity and allogenic transplantation inheres the risk of transmission of diseases or rejection [115, 116].

    In the last two decades, regenerative medicine approaches have been extensively studied to improve bone healing, or even generate functional bone tissue to substitute lost bone. Many in vitro studies were performed to investigate applicability of different stem cell types for bone regeneration. Here, promising capacity for differentiating towards bone cells, enhancing bone healing and vascularization could be proven for embryonic stem cells and different adult mesenchymal stem cells. However, due to the ethical and safety concerns mentioned above, only adult stem cells are presently taken into consideration for therapeutic applications [63]. Here, mesenchymal stem cells presently seem to be the most promising candidates for bone regeneration, due to their excellent osteogenic differentiation capacity [69].

    In vitro trials found out that MSC strongly promote angiogenesis by paracrine factors after mechanical stimulation, as occurring during fracture healing [117], which makes MSC more interesting for bone regeneration. This paracrine enhancement of angiogenesis in bone regeneration could also be confirmed in animal models in vivo [118].

    The capacity of mesenchymal stem cells for homing to injured tissues known from other fields was also demonstrated for fractures. Here, mesenchymal stem cells showed migration towards the fracture site after systemic application in a mouse model. The study further revealed that the cells enriched there and participated in fracture healing by paracrine induction of tissue healing, reduction of systemic and local inflammation and differentiating into bone cells [74]. However, the majority of the stem cells were trapped in the lungs after systemic application, thus making local application more practicable for bone regeneration [119].

    Different groups achieved to compose small bone-like tissue constructs in vitro, by composing MSC with a variety of different biomaterials. Implanted into animals, several of these constructs survived in vivo [120]. However, researchers did not succeed in composing vital bone pieces in larger volumes, or even whole bones. This is due to the diffusion tract being larger than 200 μm. Beyond 200 μm, diffusion is not sufficient for providing cells with oxygen and nutrients. Therefore, functional vascularization is a prerequisite for survival of such solid tissues. Up to now, the problem of vascularization in tissue engineering is not yet solved, inhibiting the translation of tissue engineering methods into the clinic [43].

    Nonetheless, regenerative medicine for bone healing has reached the patient in form of cell therapy approaches to treat localized bone defects or systemic diseases of the skeleton [39]. Here, autologous bone marrow or autologous mesenchymal stem cells was successfully implanted in a number of patients to enhance fracture/osteotomy healing, fill bone defects, treat pseudarthrosis, bone cysts, osteonecrosis, or enhance spinal fusion. Relevant clinical applications are summarized in Table 1.

    tab1
    Table 1: Clinical applications of mesenchymal stem cells in bone regeneration.

    6. Conclusions

    Current data provides a number of interesting approaches to treat musculoskeletal pathologies with the support of mesenchymal stem cells. But considering the limited, partially only preclinical data we believe that a standardized clinical application will take at least an additional 5 to 10 years. In order to realize the full therapeutic potential of stem cells, a number of open questions has to be to be answered. Besides the necessity of establishing further data about native stem cell function and pathways, basic research in the understanding of native tendon, bone, and cartilage regeneration also has to be continued. Especially signal pathways have to be understood because single-MSC application might be insufficient or only partially sufficient without the adequate signal for inducing tissue regeneration. The regenerated tissue also has to provide the appropriate 3-dimensional structure including production of extracellular matrix and biomechanical behavior according to native tissue. Therefore, tissue engineering will play an important role in the next years. In the near future, an interdisciplinary approach with biologists, bioengineers, and clinicians will be essential to achieve the clinical application of mesenchymal stem cells.

     

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