Mesenchymal Stem Cells for Osteochondral Tissue Engineering

Abstract

Mesenchymal stem cells (MSC) are of major interest in regenerative medicine, as they are easily harvested from a variety of sources (including bone marrow and fat aspirates) and they are able to form a range of mesenchymal tissues, in vitro and in vivo. We focus here on the use of MSCs for engineering of cartilage, bone, and complex osteochondral tissue constructs, using protocols that replicate some aspects of natural mesodermal development. For engineering of human bone, we discuss some of the current advances, and highlight the use of perfusion bioreactors for supporting anatomically exact human bone grafts. For engineering of human cartilage, we discuss the limitations of current approaches, and highlight engineering of stratified, mechanically functional human cartilage interfaced with bone by mesenchymal condensation of MSCs. Taken together, current advances enable engineering of physiologically relevant bone, cartilage and osteochondral composites, and physiologically relevant studies of osteochondral development and disease.

Keywords: Anatomically shaped grafts; Bioreactor; Bone; Cartilage; Regenerative medicine.

Figure 1. Tissue engineering of anatomically shaped bone grafts

(A–C) Scaffold preparation. (A, B) Clinical CT images were used to obtain high-resolution digital data for the reconstruction of the exact geometry of human temporomandibular joint (TMJ) condyles. (C) These data were incorporated into the MasterCAM software to machine TMJ-shaped scaffolds from fully decellularized trabecular bone. (D) A photograph illustrating the complex geometry of the final scaffolds that appear markedly different in each projection. (E) The scaffolds were seeded in a stirred suspension of hMSC, using 3 million cells per scaffold (~1 cm³ volume), pre-cultured for 1 week to allow cell attachment, and cultured with perfusion through the cell-seeded scaffold for an additional 4 weeks. (F) A photograph of the perfusion bioreactor used to cultivate anatomically shaped grafts in vitro. (G–I) Key steps in the bioreactor assembly. Images are reproduced with permission from reference (48).

Figure 2. Effects of perfusion on bone formation in vitro

(1) Computational models of medium flow through TMJ constructs during bioreactor cultivation. (1A) Color-coded velocity vectors indicate the magnitude and direction of flow through the entire construct based on experimentally measured parameters. (1B) Construct is digitally sectioned, and the color-coded contours are used to indicate the magnitude of flow in the inner regions. (2A–H) Bone formation was markedly enhanced by perfusion, in a manner dependent on the fluid flow pattern. (2A–D) Constructs cultured under static conditions. (2E–H) Constructs cultured with medium perfusion. (2A, E) Trichrome staining of the entire cross-section of scaffolds showing differences in the new matrix distribution (red) compared with the original scaffold (green) for the static (2A) and perfused (2E) culture groups. (2B, F) Major differences in osteoid formation (arrows) in the central regions of constructs cultured statically (2B) and in perfusion (2F). (2C, D, G, H) SEM images of the central construct regions. (2C, D) Statically cultured constructs exhibit empty pore spaces and loosely packed cells. (2G, H) Constructs cultured in perfusion demonstrate the formation of dense and confluent lamellae of bone tissue that fill entire pore spaces. (Scale bars - 2A, E: 5 mm; 2B, C, F, G: 1 mm; 2D, H: 500 μm.) (3A–C) Architecture of the mineralized bone matrix developed over time and in a manner dependent on culture conditions. The reconstructions of 3D μCT images demonstrate the changes in pore structure (relative to the initial state) that were evident at the end of the 5^th week of cultivation. (Scale bar: 5 mm.) Images reproduced with permission from reference (48)

Figure 3. In vitro formation of physiologically stratified, stiff and frictionless human cartilage interfaced with a bone substrate

Cartilage was formed from condensed mesenchymal bodies (CMBs) press fit onto a bone substrate, and cultured in vitro, as reported by Bhumiratana et al (71). (A–B) CMBs were press-fitted within molds on a decellularized bone scaffold (DCB), forming cartilage after 5 weeks of chondrogenic induction. Histological and immunohistochemical analysis showing representative stains of (C–F) H&E, (G–J) Alcian Blue for GAG, (K–N) collagen type II, (O–R) lubricin, (S–V) collagen type I, and (W–Z) collagen type X. (Scale bar: 500 μm in low-magnification images, 50 μm in high-magnification images.)

Figure 4. Approach to assembling osteochondral composites

The schematic shows one of the scaffolds and cell based strategies for engineering osteochondral composites discussed here, proposed by Martin et al (72).

Figure 5. Osteochondral composites obtained by combining pre-grown cartilage and bone

The panel shows osteochondral constructs cultured for 10 weeks following the assembly of cartilage and bone regions. (A–C) H&E for the cells and matrix of the cartilage layer (A), sharply demarcated transition zone (B, arrows), and an osseous layer (C); (D–F) alcian blue, for proteoglycan that is strongly positive in the cartilage layer (D), diminishingly positive in the transition zone (E, arrows), and negative in the osseous layer (F); (G–I) alizarin red staining, for mineralization that was negative in the cartilage layer (G), and strongly positive in the transition zone (H, arrows) and the osseous layer (I). CL: cartilage layer; OL: osseous layer. Scale bars: 80 μm. Reproduced with permission from Tuli et al (78).