Cartilage tissue engineering using dermis isolated adult stem cells: the use of hypoxia during expansion versus chondrogenic differentiation.

Dermis isolated adult stem (DIAS) cells, a subpopulation of dermis cells capable of chondrogenic differentiation in the presence of cartilage extracellular matrix, are a promising source of autologous cells for tissue engineering. Hypoxia, through known mechanisms, has profound effects on in vitro chondrogenesis of mesenchymal stem cells and could be used to improve the expansion and differentiation processes for DIAS cells. The objective of this study was to build upon the mechanistic knowledge of hypoxia and translate it to tissue engineering applications to enhance chondrogenic differentiation of DIAS cells through exposure to hypoxic conditions (5% O2) during expansion and/or differentiation. DIAS cells were isolated and expanded in hypoxic (5% O2) or normoxic (20% O2) conditions, then differentiated for 2 weeks in micromass culture on chondroitin sulfate-coated surfaces in both environments. Monolayer cells were examined for proliferation rate and colony forming efficiency. Micromasses were assessed for cellular, biochemical, and histological properties. Differentiation in hypoxic conditions following normoxic expansion increased per cell production of collagen type II 2.3 fold and glycosaminoglycans 1.2 fold relative to continuous normoxic culture (p<0.0001). Groups expanded in hypoxia produced 51% more collagen and 23% more GAGs than those expanded in normoxia (p<0.0001). Hypoxia also limited cell proliferation in monolayer and in 3D culture. Collectively, these data show hypoxic differentiation following normoxic expansion significantly enhances chondrogenic differentiation of DIAS cells, improving the potential utility of these cells for cartilage engineering

Collagen accumulation during micromass culture.

<p>Hypoxic expansion led to increased total collagen accumulation (A) and collagen accumulation per cell (C) during differentiation. Overall collagen type II production was greatest in the NH group (B) as was collagen type II production per cell (D) and relative to total collagen production (E). Data are mean ± S.D. Groups not connected by letters are significantly different. Significance defined as <i>p</i><0.05.</p

Histological sections of micromasses after 2 weeks.

<p>Collagen type I staining was strongest for HH (first row), while collagen type II staining was strongest for NH (second row). All groups stained positive for collagen (third row).</p

Hypoxia limits proliferation during 3D culture.

<p>Cells were seeded at 2×10⁵ per micromass (dashed line). Data are mean ± S.D. Groups not connected by letters are significantly different with <i>p</i><0.05.</p

Injectable and compression-resistant low-viscosity polymer/ceramic composite carriers for rhBMP-2 in a rabbit model of posterolateral fusion: a pilot study

Abstract Background The challenging biological and mechanical environment of posterolateral fusion (PLF) requires a carrier that spans the transverse processes and resists the compressive forces of the posterior musculature. The less traumatic posterolateral approach enabled by minimally invasive surgical techniques has prompted investigations into alternative rhBMP-2 carriers that are injectable, settable, and compression-resistant. In this pilot study, we investigated injectable low-viscosity (LV) polymer/composite bone grafts as compression-resistant carriers for rhBMP-2 in a single-level rabbit PLF model. Methods LV grafts were augmented with ceramic microparticles: (1) hydrolytically degradable bioactive glass (BG), or (2) cell-degradable 85% β-tricalcium phosphate/15% hydroxyapatite (CM). Material properties, such as pore size, viscosity, working time, and bulk modulus upon curing, were measured for each LV polymer/ceramic material. An in vivo model of posterolateral fusion in a rabbit was used to assess the grafts’ capability to encourage spinal fusion. Results These materials maintained a working time between 9.6 and 10.3 min, with a final bulk modulus between 1.2 and 3.1 MPa. The LV polymer/composite bone grafts released 55% of their rhBMP-2 over a 14-day period. As assessed by manual palpation in vivo, fusion was achieved in all (n = 3) animals treated with LV/BG or LV/CM carriers incorporating 430 μg rhBMP-2/ml. Images of μCT and histological sections revealed evidence of bone fusion near the transverse processes. Conclusion This study highlights the potential of LV grafts as injectable and compression-resistant rhBMP-2 carriers for posterolateral spinal fusion