Musculoskeletal injuries are a major financial burden to the U.S. health system and the most common cause of long-term pain and physical disabilities. More than 33 million injuries are reported in the United States annually; nearly half involve soft tissues such as cartilage, tendon, and ligament.
Unlike bone, which has an intrinsically high healing capacity, soft tissues have a limited capacity for self-repair. As a result, scar tissue forms, which has structural and functional characteristics that are inferior to the original tissue. The emerging field of regenerative medicine aims to overcome the tissue scarring process by providing the required elements (cells, inductive molecules, and local environment) to promote true tissue regeneration.
Tissue engineering is a multidisciplinary field that combines engineering approaches with biologic knowledge to recreate embryonic tissue development, thus providing biologic substitutes for repair or regeneration of injured tissues. Tissue engineering techniques classically involve the use of scaffolds that serve as physical templates for the matrix-forming cells to attach and orient, guided by molecular cues that dictate their phenotypic characteristics.
Historically, primary cells (such as osteoblasts or chondrocytes) have been used as building blocks for tissue regeneration. However, their use poses several limitations such as the morbidity associated with their harvesting procedure, the typical low numbers that can be obtained, their high phenotypic instability, and the intrinsic difficulties of their ex vivo manipulation.
Stem cells, on the other hand, hold great promise for skeletal tissue regeneration given their easy access, unlimited supply, expanding behavior, multipotential differentiation capacity, and recently described abilities to deliver instructions and modulate immune system reactions. When subjected to specific culture conditions, pluripotent mesenchymal stem cells (MSCs) have the ability to differentiate into various mesodermal cell lineages including osteoblasts, chondrocytes, and myocytes.
MSCs can be isolated from various tissues such as bone marrow, adipose tissue, skeletal muscle, synovium, placenta, teeth, and others. Although the phenotypical characteristics of MSCs isolated from distinct tissues may vary, they share the following common principles:
- They reside in situ as perivascular cells (pericytes).
- They retain the multipotential differentiation capacity in vitro after isolation.
As progenitors for skeletal tissues, cells, MSCs have been largely used to regenerate bone, cartilage, meniscus, intervertebral disk, tendon, and ligament with varying success. Regardless of the tissue in question, the following common issues need to be addressed when designing regenerative strategies for clinical purposes: cell source, expanding potential and generation of therapeutic-size cell population at a human scale, type of scaffold used, and in vitro preconditioning (pre-implantation optimization).
Bone formation can be achieved with acellular osteoconductive scaffolds, with or without incorporated slow-released osteoinductive molecules. The presence of osteogenic progenitor cells significantly improves clinical outcomes, especially for large bone segmental defects in both animal models and humans.
Osteogenic differentiation capabilities have been documented in vivo and in vitro with MSCs isolated primarily from bone marrow, adipose tissue, and skeletal muscle. A recently proposed alternative is the use of the nonadipocyte component of the adipose tissue, named stromal vascular fraction. Although controversial, this approach deserves more basic research, as well as preclinical and clinical documentation, given its potential advantage to easily access large quantities of osteoprogenitors and endothelial cell progenitors. These endothelial progenitors would initiate a robust and more uniform revascularization process within the implanted material—a critical step in the bone formation program.
Various preclinical models and clinical applications have been described using MSCs loaded into various different scaffolds (Table 1). These osteoconductive matrices promote bone ingrowth and may serve as reservoirs for control-released osteoinductive molecules, the best studied of which is recombinant human bone morphogenetic protein-2 (rhBMP-2).
Becaue the recombinant proteins have a restricted kinetic profile, various groups have used genetically modified MSCs to generate a more sustained presence of osteoinductive molecules. These genetically modified MSCs overexpress different inductive cues and have shown promising preclinical results in terms of bone formation and healing response. This gene therapy approach also has been used to promote the expression of angiogenic molecules such as vascular endothelial growth factor (VEGF), based on the known association between bone formation and vascular presence.
A new perspective for bone repair is based on the novel concept of osteoprogenitors residing as pericytes in the bone marrow vasculature. This intricate physical association between blood vessels and bone cell progenitors reinforces the pivotal role of the vasculature in bone formation and provides potential mechanistic explanations for the action of certain osteoinductors such as platelet-derived growth factor-B (PDGF-B). Related to this hypothesis, during bone formation (in both development and repair), the pericytes can be dissociated from their perivascular location and multiplied in the presence of PDGF-B. Then, specific inductors such as BMPs and Wnt signaling molecules (modulated by PDGF-B signaling) can be used to drive osteogenic differentiation.
Although bone has a high reparative capacity, articular cartilage exhibits a poor intrinsic healing ability due to structural and biologic characteristics. Articular cartilage has a complex, layered and avascular tri-dimensional structure, with an extracellular matrix (ECM) made by a few highly specialized cells (chondrocytes), which typically divide very slowly. This disposition “isolates” the tissue from its surroundings, compromising normal reparative attempts.
Current cartilage treatment options can be divided into stimulation and replacement strategies. Stimulation techniques attempt to bring mesenchymal progenitors from the marrow to differentiate and repopulate the defect. Although these techniques have shown good short-term results, the repair tissue eventually fails due to the inferior structural and mechanical qualities of the resulting fibrocartilage.
Replacement, on the other hand, tries to fill the defect with a native tissue obtained from nearby healthy locations. Long-term complications, donor site-associated morbidity, and the scarcity of replacement tissue are the major disadvantages of the autologous component of this technique. The use of allografts, however, incorporates potential immunological rejection concerns.
Cell-based therapy for cartilage repair has gained acceptance with the promising results obtained with autologous chondrocyte implantation. Disadvantages of this approach include the limited supply of cells and the associated donor site morbidity. Alternatively, the use of MSCs has been proposed and extensively studied, due to their chondrogenic differentiation capacity, large numbers, expandable potential, and potential for building tissue through a process similar to embryonic development.
Recent evidence in animal models suggests that MSCs used to repair osteochondral defects rapidly differentiate into cartilaginous ECM-producing chondrocytes in the chondral part of the implant, while filling the subchondral space with highly vascularized bone, fully integrated with the host tissue. Despite the apparent success, the implant showed abnormal remodeling at long-term, compromising the ECM quantity and quality.
New methods to stimulate MSCs and generate a more durable implant can be broadly divided into pre- and postimplantation strategies. In vitro optimization techniques (pre-implantation) involve the use of specific scaffold materials that promote cell survival and induce cell differentiation; growth factors to manipulate cell proliferation, differentiation, ECM production, and cellular hypertrophic terminal differentiation; adjusted culture conditions to mimic the native environment (use of flow stimulation in a bioreactor); and genetically engineered MSCs to express specific targets (ie, transforming growth factor-beta-1 and insulin-like growth factor-I).
After the graft is implanted, anabolic factors can be injected intra-articularly to modulate the implant behavior in situ. This approach can also be used without grafts, to stimulate the endogenous repair capacity and/or to modify the clinical course of the causing disease, as has been shown with fibroblast growth factor (FGF) 18 in osteoarthritis (OA).
The major issues with engineered implants to repair cartilage defects that are currently under investigation are related to the in situ integration with the host tissue and the mechanical properties of the regenerated tissue. Therapeutically, efforts are concentrated on developing osteochondral plugs to treat localized defects and on creating larger constructs for total joint resurfacing to manage conditions such as OA.
Other skeletal tissues
The basic tissue engineering principles discussed above also apply for tissues such as meniscus, ligament, and tendon. The idea is to restore the architecture of the tissue with MSC-loaded scaffolds, modifying the regenerative response by preconditioning the construct before implantation (through mechanical and growth factor stimulation), and modulating its behavior after implantation. Given the critical role of certain growth factors controlling the repair response, the generation of engineered MSCs to express specific molecules (such as FGFs, EGF, PDGF, and BMPs) as part of a gene therapy approach has been encouraging, especially in meniscal repair.
Finally, previously unrecognized functions of MSCs, related to their trophic and immunomodulatory capacities, are now the subject of intense clinical investigation. By secreting large quantities of various cytokines and growth factors and modulating the local immune response, MSCs can have a significant impact on tissue inflammation, healing, and potentially rejection of allografts. Consequently, new strategies for skeletal tissue regeneration must include these two functions as part of the design rationale. Currently, 35 clinical trials using MSCs to treat various skeletal conditions are underway worldwide.
Diego Correa, MD, MSc, PhD is a senior research associate at the Skeletal Research Center, Case Western Reserve University. Victor M. Goldberg, MD, is professor of orthopaedic surgery at Case Western Reserve University and University Hospitals of Cleveland.
Disclosure information: Dr. Correa—Sciencia Stem Cell consulting; National Institutes of Health (NIH); Dr. Goldberg—Osteotech; AstraZeneca; TissueLink; NIH; Sultzer/Zimmer; Elsevier; Journal of Bone and Joint Surgery–American; Journal of Orthopaedic Research; Clinical Orthopaedics and Related Research; Osteoarthritis and Cartilage; AAOS Now; OASRI; Bioinnovations Institute
- Because they can be harvested in large quantities, expanded ex vivo, and exhibit multipotential differentiation capacity, mesenchymal stem cells (MSCs) have been used to regenerate bone, cartilage, meniscus, intervertebral disk, tendon, and ligament with varying success.
- A new perspective for bone repair is based on the novel concept of osteoprogenitors residing as perivascular cells (pericytes) in the bone marrow vasculature.
- The use of MSCs for cartilage repair must address integration with the host tissue and the mechanical properties of the regenerated tissue.
- By secreting large quantities of various cytokines and growth factors and modulating the local immune response, MSCs can have a significant impact on tissue inflammation, healing, and, potentially, allograft rejection.