Towards engineering distinct multi-lamellated outer and inner annulus fibrosus tissues.

The annulus fibrosus (AF) of the intervertebral disc (IVD) has a zonal distribution of phenotypically distinct cells. The outer AF (OAF) cells produce an extracellular matrix (ECM) rich in type I collagen with little proteoglycans, whereas the ECM of the inner AF (IAF) has abundant type II collagen and proteoglycans. The inhomogeneous distribution of the ECM in the AF may reflect the complex mechanical forces that the IVD experiences. A bioengineered AF tissue should recapitulate both the inner and outer zones in order to have proper functionality. The aim of this study is to generate multi-lamellated OAF and IAF tissues with ECM compositions that resemble their zonal origin using polycarbonate urethane (PU) scaffolds. It was observed that supplementation of the media with insulin-transferrin-selenium (ITS) and proline yielded tissues with good cellularity. However, IAF cells accumulated only type I collagen, similar to OAF cells. Addition of dexamethasone and sodium pyruvate induced the accumulation of IAF tissues rich in type II collagen and aggrecan, without altering the accumulation of type I collagen in OAF tissues. Dexamethasone stimulated mitochondrial membrane potential in both tissues in the presence of sodium pyruvate, suggesting a relationship between the mitochondrial aerobic respiratory state and dexamethasone signalling during the in vitro-tissue formation by OAF and IAF cells. Inhibition of the glucocorticoid receptor blocked the stimulation of mitochondrial membrane potentials and type II collagen accumulation. In summary, biologically distinct multi-lamellated OAF and IAF tissues can be generated, which will facilitate advancement towards the goal of engineering a biological IVD replacement. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1346-1355, 2018.

Inorganic polyphosphates enhances nucleus pulposus tissue formation in vitro.

Disc degeneration is associated with low back pain for which currently there is no optimal therapy so there is a great need to identify new treatment approaches. Inorganic polyphosphates (polyP) are linear polymers of orthophosphate units varying in chain length and present in many cell types. As polyP has anabolic effects on chondrocytes, we hypothesized that polyP treatment would enhance matrix accumulation by nucleus pulposus (NP) cells. NP cells isolated from bovine caudal discs were grown in 3D culture under normoxic or in select experiments under hypoxic conditions, in the presence or absence of various concentrations and sizes of polyP. Gene expression was determined using RT-PCR. Matrix accumulation was quantified by measuring proteoglycan and collagen contents. DAPI fluorescence shift was used to stain for polyP in tissue. DAPI staining showed polyP present predominantly in the pericellular region of in vitro formed tissue. PolyP treatment enhanced matrix accumulation in a concentration and chain length dependant manner. NP cells exposed to polyP-22 (22 phosphate units length) showed an increase in gene expression of aggrecan, Collagen II, Sox 9, and MMP-13 which was maintained for the 14 days of culture. This suggests that polyP may enhance NP tissue formation in vitro by upregulating the expression of matrix genes. As polyP enhances proteoglycan accumulation even under hypoxic conditions, this raises the possibility that polyP may be a novel treatment to induce NP regeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:41-50, 2017.

Supplementation with platelet-rich plasma improves the in vitro formation of tissue-engineered cartilage with enhanced mechanical properties.

PURPOSE: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. METHODS: Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). RESULTS: Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 µg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 µg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 µg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 µg/mg), 20% FBS (7.6 ± 0.9 µg/mg; P = .37), or 20% PPP (6.4 ± 1 µg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 µg/mg), 20% FBS (9.3 ± 2.5 µg/mg; P = .99), or 20% PPP (7.2 ± 1.3 µg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. CONCLUSIONS: The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. CLINICAL RELEVANCE: Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.