Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells.

INTRODUCTION: Notochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype. METHODS: Human MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGFß-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT. RESULTS: Notochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group. CONCLUSIONS: NCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions in which media was generated (cells in alginate versus cells in their native tissue) so that further mechanistic studies optimizing culture conditions and defining important NC secreted factors are required. Matricellular proteins, such as clusterin and tenascin, are likely to be important to optimize differentiation of MSCs for maximum GAG production and reduced collagen fiber expression.

Combined use of designed scaffolds and adenoviral gene therapy for skeletal tissue engineering.

While tissue engineering remains the most researched alternative to conventional therapies for repair and regeneration, how to optimally combine two of the most promising techniques, designed solid scaffolds and localized gene therapy, is largely unknown. We have conducted a systematic screening of several variables that may affect generation of bone via adenoviral gene therapy vector delivery, on image-based designed and solid freeform-fabricated scaffolds. These variables included: gene therapy type (ex vivo or in vivo); scaffold base material (sintered hydroxyapatite or a polypropylene fumarate/ tricalcium phosphate (PPF/TCP) composite), secondary carrier used to attach the biofactor to the scaffold (fibrin gel or a poly-lactic acid sponge), and scaffold pores size (300 or 800 microm). The in vivo formation of bone following implantation of these scaffolds was then analyzed. Gene therapy method had the largest effect, with ex vivo gene therapy yielding significant amounts of bone on nearly all the implants and in vivo gene therapy failing to produce any bone on most implants. Secondary carrier was the next most important variable, with fibrin gel consistently producing bone encompassing the implants and producing 2-4 times as much bone as the polymer sponge, which triggered only isolated bone growth. Though both scaffold base materials allowed bone growth, hydoxyapatite scaffolds generated twice as much bone as PPF/TCP scaffolds. The pore sizes tested had no significant effect on tissue generation.

Functional bone engineering using ex vivo gene therapy and topology-optimized, biodegradable polymer composite scaffolds.

Bone tissue engineering could provide an alternative to conventional treatments for fracture nonunion, spinal fusion, joint replacement, and pathological loss of bone. However, this approach will require a biocompatible matrix to allow progenitor cell delivery and support tissue invasion. The construct must also support physiological loads as it degrades to allow the regenerated tissue to bear an increasing load. To meet these complex requirements, we have employed topology-optimized design and solid free-form fabrication to manufacture biodegradable poly(propylene fumarate)/beta-tricalcium phosphate composites. These scaffolds were seeded with primary human fibroblasts transduced with an adenovirus expressing bone morphogenetic protein-7 and implanted subcutaneously in mice. Specimens were evaluated by microcomputed tomography, compressive testing, and histological staining. New bone was localized on the scaffold surface and closely followed its designed contours. Furthermore, the total stiffness of the constructs was retained for up to 12 weeks after implantation, as scaffold degradation and tissue invasion took place.

Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering.

Polycaprolactone (PCL) is a bioresorbable polymer with potential applications for bone and cartilage repair. In this work, porous PCL scaffolds were computationally designed and then fabricated via selective laser sintering (SLS), a rapid prototyping technique. The microstructure and mechanical properties of the fabricated scaffolds were assessed and compared to the designed porous architectures and computationally predicted properties. Scaffolds were then seeded with bone morphogenetic protein-7 (BMP-7) transduced fibroblasts and implanted subcutaneously to evaluate biological properties and to demonstrate tissue in-growth. The work done illustrates the ability to design and fabricate PCL scaffolds with porous architecture that have sufficient mechanical properties for bone tissue engineering applications using SLS. Compressive modulus and yield strength values ranged from 52 to 67 MPa and 2.0 to 3.2 Mpa, respectively, lying within the lower range of properties reported for human trabecular bone. Finite element analysis (FEA) results showed that mechanical properties of scaffold designs and of fabricated scaffolds can be computationally predicted. Histological evaluation and micro-computed tomography (microCT) analysis of implanted scaffolds showed that bone can be generated in vivo. Finally, to demonstrate the clinical application of this technology, we designed and fabricated a prototype mandibular condyle scaffold based on an actual pig condyle. The integration of scaffold computational design and free-form fabrication techniques presented here could prove highly useful for the construction of scaffolds that have anatomy specific exterior architecture derived from patient CT or MRI data and an interior porous architecture derived from computational design optimization.

Engineered osteochondral grafts using biphasic composite solid free-form fabricated scaffolds.

Tissue engineering has provided an alternative to traditional strategies to repair cartilage damaged by injury or degenerative disease. A successful strategy to engineer osteochondral tissue will mimic the natural contour of the articulating surface, achieve native mechanical properties and functional load-bearing ability, and lead to integration with host cartilage and underlying subchondral bone. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match articular geometry. The objective of this study was to utilize materials and biological factors in an integrated approach to regenerate a multitissue interface. Biphasic composite scaffolds manufactured by IBD and SFF fabrication were used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-L-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein 7 (BMP-7) in the ceramic phase and fully differentiated chondrocytes in the polymeric phase. After subcutaneous implantation into mice, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects.