The progressive increase in life expectancy within the last century has led to the appearance of novel health related problems, some of those within the musculoskeletal field. Among the latter, one can find diseases such as osteoporosis, rheumatoid arthritis and bone cancer, just to mention some of the most relevant. Other related problems are those that arise from serious injuries, often leading to non-recoverable critical size defects. The therapies currently used to treat this type of diseases/injuries are based on the use of pharmaceutical agents, auto/allotransplant and synthetic materials. However, such solutions present a number of inconveniences and therefore, there is a constant search for novel therapeutic solutions. The appearance of a novel field of science called tissue engineering brought some hope for the solution of the above mentioned problems. In this field, it is believed that by combining a 3D porous template–scaffold–with an adequate cell population, with osteo or chondrogenic potential, it will be possible to develop bone and cartilage tissue equivalents that when implanted in vivo, could lead to the total regeneration of the affected area.
This ideal cell population should have a series of properties, namely a high osteo and chondrogenic potential and at the same time, should be easily expandable and maintained in cultures for long periods of time. Due to its natural and intrinsic properties, stem cells are one of the best available cell types. However, after this sentence, the readers may ask, “Which Stem Cells “. During the last 10/15 years, the scientific community witnessed and reported the appearance of several sources of stem cells with both osteo and chondrogenic potential. Different sources of adult stem cells (bone marrow, periosteum, adipose tissue, skeletal muscle and umbilical cord) for bone and cartilage regenerative medicine are reported.
Bone
The regeneration of bone is a key issue at the forefront of current tissue engineering applications, owing to the ease of use and accessibility of osteoprogenitor cells. The molecular mechanisms of human MSC regulation and the importance of specific growth factors during the different stages of osteogenic differentiation are subjects of intensive investigation. The use of natural and synthetic biomaterials as carriers for MSC delivery has shown increasing promise for orthopaedic therapeutic applications, especially bone formation. Recent advances in the field of biomaterials have led to a transition from nonporous, biologically inert materials to more porous, osteoconductive biomaterials, and, in particular, the use of cell-matrix composites. A number of delivery vehicles have been successfully used in cell-matrix composites in vivo, such as porous ceramics of hydroxyapatite and -tricalcium phosphate loaded with autologous MSCs. These constructs were capable of healing critical-sized segmental bone defects not capable of being healed by resident cells or by the addition of the osteoconductive device alone. An in vitro study comparing the biodegradable polymers poly-L-lactide (PLA) and poly-L-lactide-co-glycolide (PLGA) on the basis of adherence and proliferation of seeded trabecular-bone-derived osteoprogenitor cells showed that PLGA was the better substrate for the attachment and subsequent osteogenic differentiation of these progenitor cells.
Mesenchymal stem cell-based bone tissue engineering
The regeneration of bone is a key issue at the forefront of current tissue engineering applications, owing to the ease of use and accessibility of osteoprogenitor cells.
Molecular regulation of osteogenic differentiation
The molecular mechanisms of human Mesenchymal stem cell (MSC) regulation and the importance of specific growth factors during the different stages of osteogenic differentiation are subjects of intensive investigation. The induction of MSC osteogenesis is a highly programmed process, best illustrated in vitro. Treatment with the synthetic glucocorticoid dexamethasone stimulates MSC proliferation and supports osteogenic lineage differentiation. Organic phosphates, such as ?-glycerophosphate, also support osteogenesis by playing a role in the mineralization and modulation of osteoblast activities. Free phosphates can induce the mRNA and protein expression of osteogenic markers such as osteopontin, and these phosphates have known effects on the production and nuclear export of a key osteogenesis regulatory gene, Cbfa1 (core binding factor alpha1). Other supplements, such as ascorbic acid phosphate and 1,25-dihydroxyvitamin D3, are commonly used for osteogenic induction, with the latter involved in increasing alkaline phosphatase activity in osteogenic cultures and promoting the production of osteocalcin. In addition to established supplements, members of the bone morphogenetic protein (BMP) family of growth factors are also routinely used for osteoinduction. BMP-2 alone appears to increase bone nodule formation and the calcium content of osteogenic cultures in vitro, while concomitant application of BMP-2 and basic fibroblast growth factor increases MSC osteogenesis both in vivo and in vitro. A number of signaling pathways have been shown to participate in MSC osteogenesis. The secreted signaling proteins known as Wnts have been implicated in various differentiation programs, including osteogenesis.
Bone tissue engineering
The use of natural and synthetic biomaterials as carriers for MSC delivery has shown increasing promise for orthopaedic therapeutic applications, especially bone formation. Recent advances in the field of biomaterials have led to a transition from nonporous, biologically inert materials to more porous, osteoconductive biomaterials, and, in particular, the use of cell-matrix composites. A number of delivery vehicles have been successfully used in cell-matrix composites in vivo, such as porous ceramics of hydroxyapatite and ?-tricalcium phosphate loaded with autologous MSCs. These constructs were capable of healing critical-sized segmental bone defects not capable of being healed by resident cells or by the addition of the osteoconductive device alone. An in vitro study comparing the biodegradable polymers poly-L-lactide (PLA) and poly-L-lactide-co-glycolide (PLGA) on the basis of adherence and proliferation of seeded trabecular-bone-derived osteoprogenitor cells showed that PLGA was the better substrate for the attachment and subsequent osteogenic differentiation of these progenitor cells.
Mesenchymal stem cell-based cartilage tissue engineering
Articular cartilage functions to provide uncompromised movement by minimizing friction between joints and allows load bearing through distribution of and resistance to compressive forces, but possesses very limited potential for healing. Joint pain is a major cause of disability, which most often results from damage to the articular cartilage by trauma or degenerative joint diseases such as primary osteoarthritis. Current treatment methods for restoration of function due to articular cartilage damage, other than total joint arthroplasty, include autografting, allografting, periosteal and perichondrial grafting, stimulation of intrinsic regeneration by intentionally drilling full-thickness defects, pharmacological intervention, and, finally, autologous cell transplantation such as the periosteal flap technique. Despite such advances, cartilage damage often cannot be repaired to a fully functional normal state, or the procedures have higher failure rates in younger patients.
Cartilage repair and regeneration by stem cell-based tissue engineering could be of enormous therapeutic and economic potential benefit for an aging population. A potential resolution of this disease state is the regeneration of cartilage tissue using autologous MSCs, thereby obviating any donor-site morbidity as is seen with current repair methods. However, to use stem cells effectively, requiring an understanding of the mechanisms responsible for the generation, maintenance, and particularly the regeneration of cartilage tissues; their natural environment must be understood in order to expand them in vitro without compromising their multilineage potential and their specific differentiation program. The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration.
Molecular regulation of chondrogenic differentiation
The induction of chondrogenesis in MSCs depends on the coordinated activities of many factors, including parameters such as cell density, cell adhesion, and growth factors. For example, culture conditions conducive for chondrogenic induction of MSCs require high-density pelleting and growth in serum-free medium containing specific growth factors and supplements. The TGF- superfamily of proteins and their members, such as the bone morphogenetic proteins (BMPs), are well-established regulatory factors in chondrogenesis. TGF- 1 was initially used for in vitro culture and can induce chondrogenesis under these conditions, although TGF- 3 has recently been shown to induce a more rapid and thorough expression of chondrogenic markers. Another TGF- family member, BMP-6, appears to increase the size and weight of pellet cultures and to increase the amount of matrix proteoglycan produced. BMP-2 and BMP-9 have also been used in three-dimensional MSC culture systems, such as those seeded in the hydrogel alginate, and under these conditions can induce markers of chondrogenesis.
Similar to their role in chondrogenesis during development, the Wnt and Wnt-related family of signaling proteins are also involved in adult cartilage homeostasis. While a number of Wnts have been shown to inhibit chondrogenesis in vitro and in vivo. In humans, mutations in the Wnt-1-inducible signaling pathway protein3 (WISP-3) are associated with the autosomal recessive disorder progressive pseudorheumatoid dysplasia. Patients with this disorder present primarily with a continual loss of cartilage as they age, which is accompanied by destructive bone changes. WISP-3 is closely related to WISP-1 and WISP-2, both of which are highly expressed in Wnt-1-transformed cells. These WISP proteins are of the same family of proteins as connective tissue growth factor, which is regulated by TGF- . Interestingly, WISP-3 is expressed in adult human synoviocytes and articular cartilage, and other Wnts, such as Wnt-11, are expressed in developing cartilage and are upregulated during MSC chondrogenesis, suggesting the involvement of the Wnt signaling cascade in MSC chondrogenic differentiation. Consistent with this hypothesis, Wnt family members are present in vivo in the joint and in vitro in chondrogenic pellet cultures.
Other signaling cascades involved in crosstalk with TGF- include the mitogen-activated protein kinase (MAPK) pathways. Studies have shown that activation of the p38, ERK, and JNK MAP kinases is required for the chondrogenic induction and maintenance of TGF- 1 treated trabecular-bone-derived MSC cultures. Inhibition of the individual MAP kinase pathways with specific chemical inhibitors either completely abolished or significantly reduced expression levels of cartilage-specific genes in a pattern distinct to each pathway, thus indicating that p38, ERK, and JNK are independently essential for the TGF- 1-mediated induction of chondrogenesis.
At the level of transcriptional regulation, changes in the levels of cellular binding of the transcription factors Sp-1 and AP-2 to their cognate response DNA sequences contained within the proximal promoter region of the gene of a cartilage matrix component, aggrecan, are indeed the targets of TGF- 1-induced MSC chondrogenesis. Another key factor known to play a role in chondrogenic lineage commitment and differentiation, and in the activation of cartilage-specific genes, is the transcription factor Sox9, whose mRNA levels are increased during chondrogenesis, particularly at early time points
Cartilage tissue engineering
MSC-based repair of full-thickness articular cartilage defects has been attempted in animal models, using various carrier matrices. Natural polymers such as collagen have shown promise in early applications. Using autologous MSCs dispersed in a collagen-type-I gel, Wakitani et al. succeeded in repairing full-thickness defects on the weight-bearing surface of medial femoral condyles. The regenerating cartilage was subsequently replaced by bone in a proximal-to-distal fashion until the underlying subchondral bone was completely repaired without disruption of the overlying cartilage.
Use of synthetic polymers in such applications have also been promising, in particular the -hydroxyesters PLA and PGA and their copolymer, PLGA. Recent work in our laboratory has also tested the efficacy of using such biomaterials, with modifications, in MSC-based cartilage tissue engineering. Caterson et al. recently evaluated the use of an amalgam consisting of PLA and the hydrogel alginate as a three-dimensional carrier for MSC-based cartilage formation in vitro. Alginate significantly improved cell loading and retention within the construct and maintained a round cell shape to enhance the chondrogenic differentiation of MSCs, while PLA provided appropriate mechanical support and stability to the composite culture, suggesting the amalgam as a potential candidate bioactive scaffold. We have also successfully fabricated ‘plug-like’ cartilage constructs by press-coating PLA polymer blocks onto high-density cell pellets of human MSCs treated with TGF- 1 in a chondrogenic environment. Scanning electron microscopy and histological analysis revealed spatially distinct cellular zones, with the superficial layer resembling hyaline cartilage, and immunohistochemically detectable collagen typeII and cartilage proteoglycan link protein within the extracellular matrix, suggesting the potential utility of this construct for tissue-engineered therapy of articular cartilage defects [117]. Our recent attempts to fabricate a single-unit osteochondral plug on the PLA block using press-coated cartilage followed by seeded osteoblasts, all derived from the same MSC source, have been promising (R Tuli et al., unpublished observation). Recently, Li et al. have developed a novel nanofibrous biomaterial, based on PLGA and poly- -caprolactone, by using an electrospinning process to fabricate a unique three-dimensional scaffold with structural similarity to a natural collagen network, as well as the ability to support MSC attachment, proliferation, and differentiation [[118]; Li et al., unpublished observation]. In particular, the slower degradation rate of poly- -caprolactone compared with other polyesters may make it a highly suitable candidate biomaterial for the delivery of growth factors such as TGF- 1, and the properties can be further modified by copolymerizing with other polyesters. Such constructs may be applicable for the clinical reconstruction of articular cartilage defects.
