Tissue-Engineered Composites of Anulus Fibrosus and Nucleus Pulposus for Intervertebral Disc Replacement

Abstract

In Brief Study Design. By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. Objectives. The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. Summary of Background Data. Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. Methods. Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in −70 C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. Results. The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. Conclusion. These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications. This study describes the generation of composite intervertebral discs consisting of tissue-engineered anulus fibrosus and nucleus pulposus implanted subcutaneously into athymic mice for up to 12 weeks. Implants exhibited excellent shape retention, with distinct anulus fibrosus and nucleus pulposus zones that integrated with time. Histologic and biochemical features of engineered intervertebral discs were similar to native tissue.