Introduction: By the year 2030, osteoarthritis is expected to affect 67 million Americans. Osteoarthritis commonly leads to the creation of osteochondral lesions. These lesions disrupt the unique zonal organization of osteochondral extracellular matrix, which proves vital to its function. There exists a need for tissue engineering strategies to induce the regeneration of new tissue that recapitulates this zonal organization and corresponding functionality. In the current work, a sequential freeze casting process is detailed that allows for the fabrication of multi-zonal collagen-based scaffolds that mimic three zonal features of native osteochondral extracellular matrix: the superficial zone (SZ), the transition zone (TZ), and the deep zone (DZ) morphologies, zonal pore size gradient, and zone-specific compositions of articular cartilage and subchondral bone in a single construct. To mimic the zonal structure, a multizonal scaffold has been designed through a novel sequential freezing process for osteochondral defect repair. Zonal anisotropy is created through two stages of unidirectional freezing. Zone-specific pore size is controlled through applied cooling rate and zone-specific composition is controlled by adjusting initial suspension formulations. Methods: Sequential freeze casting was performed to create multizonal collagen (Col) and collagen-hyaluronic acid (Col-HYA)/collagen-hydraxyapatite (Col-HA) composite scaffolds. For collagen-only scaffolds, 2 wt% type Ⅰ collagen was homogenized in deionized water, degased, and unidirectionally frozen. The frozen structure was flipped 90°, partially melted, and a second collagen suspension was loaded and unidirectionally frozen atop the first. Sequentially frozen scaffolds were lyophilized at -40°C for 3 days, crosslinked with 2% EDC (1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride):NHS (N-hydroxysuccinimide) (1:0.25 molar ratio), and lyophilized for another 3 days. The effect of cooling rate on zonal pore size was investigated. SEM analysis was performed to view zonal morphologies (longitudinal sections) and quantify pore sizes (transverse sections). To further mimic the zonal composition of native osteochondral extracellular matrix, HYA and HA was added to the collagen suspensions and the scaffolds were prepared using the approached described above. A Col-HYA (1.8 wt% Col, 0.2 wt% HYA) suspension was first unidirectionally frozen at 2°C/min. A second Col-HYA slurry of increased HYA and decreased Col content was frozen 90° to the first at 0.5°C/min, where a third Col-HA slurry was layered atop. Results and Discussion: SEM analysis reveals three distinct zones of collagen fiber orientation seamlessly integrated together (Figure 1). A 200-300 μm transition region of isotropic collagen integrates between two zones of unidirectionally aligned fibers, perpendicular to one another. Pore size quantification revealed a trend of increasing pore sizes through the depth of the scaffold. Zonal pore sizes were controlled by the applied cooling rate. Quicker cooling led to the formation of smaller pores, while slower cooling promoted larger pores. Fig. 1: Scanning electron micrograph of collagen-based multizonal scaffolds. SZ - superficial zone, TZ - transition zone, DZ - deep zone. Conclusions: Sequentially freeze cast multizonal collagen-based scaffolds were fabricated that mimic the zone-specific collagen fiber orientation, pore size, and composition present in native osteochondral tissue.
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