Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes.

BACKGROUND: Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N. METHODS: In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison. RESULTS: Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels. CONCLUSION: Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Evaluation of quantitative magnetic resonance imaging, biochemical and mechanical properties of trypsin-treated intervertebral discs under physiological compression loading.

PURPOSE: To investigate the influence of targeted trypsin digestion and 16 hours compression loading on MR parameters and the mechanical and biochemical properties of bovine disc segments. MATERIALS AND METHODS: Twenty-two 3-disc bovine coccygeal segments underwent compression loading for 16 hours after the nucleus pulposus (NP) of each disc was injected with a solution of trypsin or buffer. The properties of the NP and annulus fibrosus (AF) tissues of each disc were analyzed by quantitative MRI, biochemical tests, and confined compression tests. RESULTS: Loading had a significant effect on the MR properties (T(1), T(2), T(1rho), MTR, ADC) of both the NP and AF tissues. Loading had a greater effect on the MR parameters and biochemical composition of the NP than trypsin. In contrast, trypsin had a larger effect on the mechanical properties. Our data also indicated that localized trypsin injection predominantly affected the NP. T(1rho) was sensitive to loading and correlated with the water content of the NP and AF but not with their proteoglycan content. CONCLUSION: Our studies indicate that physiological loading is an important parameter to consider and that T(1rho) contributes new information in efforts to develop quantitative MRI as a noninvasive diagnostic tool to detect changes in early disc degeneration.

Quantitative magnetic resonance imaging of enzymatically induced degradation of the nucleus pulposus of intervertebral discs.

STUDY DESIGN: The structural integrity of the nucleus pulposus (NP) of intervertebral discs was targeted by enzyme-specific degradations to correlate their effects to the magnetic resonance (MR) signal. OBJECTIVE: To develop quantitative MR imaging as an accurate and noninvasive diagnostic tool to better understand and treat disc degeneration. SUMMARY OF BACKGROUND DATA: Quantitative MR analysis has been previously shown to reflect not only the disc matrix composition, but also the structural integrity of the disc matrix. Further work is required to identify the contribution of the structural integrity versus the matrix composition to the MR signal. METHODS: The bovine coccygeal NPs were injected with either enzyme or buffer, incubated at 37 degrees C as static, unloaded and closed 3-disc segments, and analyzed by a 1.5-Tesla MR scanner to measure MR parameters. RESULTS: Collagenase degradation of the NP significantly decreased the relaxation times, slightly decreased the magnetization transfer ratio, and slightly increased the apparent diffusion coefficient. Targeting the proteoglycan and/or hyaluronan integrity by trypsin and hyaluronidase did not significantly affect the MR parameters, except for an increase in the apparent diffusion coefficient of the disc after trypsin treatment. CONCLUSIONS: Our results demonstrate that changes in the structural integrity of matrix proteins can be assessed by quantitative MR.

Selective inhibition of type X collagen expression in human mesenchymal stem cell differentiation on polymer substrates surface-modified by glow discharge plasma.

Recent evidence indicates that a major drawback of current cartilage- and disc-tissue engineering is that human mesenchymal stem cells (MSCs) rapidly express type X collagen-a marker of chondrocyte hypertrophy associated with endochondral ossification. Some studies have attempted to use growth factors to inhibit type X collagen expression, but none to date has addressed the possible effect of the substratum on chondrocyte hypertrophy. Here, we sought to examine the growth and differentiation potential of human MSCs cultured on two polymer types, polypropylene and nylon-6, both of which have been surface-modified by glow discharge plasma treatment in ammonia gas. Cultures were performed for up to 14 days in Dulbecco's modified Eagle medium + 10% fetal bovine serum. Commercial polystyrene culture dishes were used as control. Reverse transcriptase-polymerase chain reaction was used to assess the expression of types I, II, and X collagens and aggrecan using gene-specific primers. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. Types I and X collagens, as well as aggrecan, were found to be constitutively expressed by human MSCs on polystyrene culture dishes. Whereas both untreated and treated nylon-6 partially inhibited type X collagen expression, treated polypropylene almost completely inhibited its expression. These results indicate that plasma-treated polypropylene or nylon-6 may be a suitable surface for inducing MSCs to a disc-like phenotype for tissue engineering of intervertebral discs in which hypertrophy is suppressed.

Biological evaluation of chitosan salts cross-linked to genipin as a cell scaffold for disk tissue engineering.

Degenerative disc disease has been implicated as a major component of spine pathology. However, although biological repair of the degenerate disk would be the ideal treatment, there is no universally accepted scaffold for tissue engineering of the intervertebral disk. To help remedy this, we investigated the gelation kinetics of various concentrations (2.5 to 10%) of two water-soluble chitosan chlorides (low molecular weight Protasan UP CL113 and high molecular weight Protasan UP CL213) and two chitosan glutamates (low molecular weight Protasan UP G113 and high molecular weight Protasan UP G213). Various concentrations (5 to 20%) of genipin, a naturally occurring cross-linking reagent used in herbal medicine and in the fabrication of food dyes, were used to prepare cross-linked chitosan hydrogels. The results show that 2.5% Protasan UP G213 cross-linked to 5% genipin was the best candidate. This formulation gelled fastest at 37 degrees C, and maintained 95% viability of encapsulated cultured disk cells. The gel did not produce an inflammatory reaction when injected subcutaneously into C57BL/6 mice and is therefore biocompatible. Most importantly, when injected into the degenerated nucleus pulposus of human cadaveric intervertebral disk, the gel flowed into the clefts without leakage. This study demonstrates that 2.5% Protasan UP G213 cross-linked to 5% genipin might be a promising scaffold for disk tissue engineering.

Value and limitations of using the bovine tail as a model for the human lumbar spine.

STUDY DESIGN: The contents of DNA, proteoglycan, type II collagen, and denatured type II collagen in the bovine coccygeal intervertebral discs were examined in situ in relation to disc level, age, and tissue region. OBJECTIVE: To determine whether bovine coccygeal discs are a suitable model to study human lumbar discs. SUMMARY OF BACKGROUND DATA: Bovine coccygeal discs have been suggested as a suitable alternative model because they are readily available, in contrast to human discs, and represent a common source of tissue in the disc field. However, it is not known whether the changes in matrix contents in bovine coccygeal discs are similar to those found in the human lumbar spine. METHODS: Intervertebral discs from bovine tails were dissected into the nucleus pulposus (NP) and anulus fibrosus (AF). Tissues were weighed and analyzed for matrix contents using specific assays. RESULTS: Similar to water content, the proteoglycan content was higher in the NP than in the AF. Water content of the bovine NP did not change with age, unlike the proteoglycan content, which decreased. type II collagen content was higher in the NP than in the AF, and both did not change overall significantly with age. The percent of denatured type II collagen decreased with age only in the NP. The DNA content did not vary with age in the AF and in the NP. CONCLUSION: Differences in matrix contents exist between the bovine coccygeal discs and the human lumbar spine. Thus, caution must be exercised when using the bovine tail as a model for the human lumbar spine in biochemical studies.

Apparent diffusion coefficient of intervertebral discs related to matrix composition and integrity.

While tremendous work has been performed to characterize degenerative disc disease through gross morphologic, biochemical, and histologic grading schemes, the development of an accurate and noninvasive diagnostic tool is required to objectively detect changes in the matrix with aging and disc degeneration. In the present study, quantitative magnetic resonance was used to determine if the quality of the nutritional supply to the intervertebral disc at various ages and levels of degeneration could be assessed through measurement of the apparent diffusion coefficients (ADCs). Modifications of the nucleus pulposus matrix content, specifically of water and glycosaminoglycan contents, with age and disc degeneration, were reflected in correlating changes in the ADCs. From unforced stepwise linear regression analyses, relations were established showing that decreases in glycosaminoglycan or water contents in the nucleus pulposus resulted in direct decreases in the ADCs. Relations obtained for the ADCs of the nucleus pulposus were direction dependent, in conformity with the anisotropic diffusion in the intervertebral discs. Changes in matrix integrity, as evidenced by the percentage of denatured collagen, were also detected in the nucleus pulposus with a low positive correlation to the ADC along the height of the disc and an inverse statistically significant regression to the ADC along the anterior to posterior axis of the disc. Correlations between the matrix content and integrity of the annulus fibrosus and its ADCs were not as evident, with only the ADC in the lateral direction of the disc of the anterior annulus fibrosus able to reflect changes in matrix content. The information obtained by the ADCs, particularly of the nucleus pulposus, can potentially be used in combination with quantitative T1, T2, and MT parameters to noninvasively obtain a quantitative assessment of the disc matrix composition and structural integrity.

A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc.

To date, there have been no reports on the effect on disc cells of the intervertebral disc (IVD) of the amino terminal peptide of link protein (DHLSDNYTLDHDRAIH) (link N) which is generated by the cleavage of human link protein by stromelysins 1 and 2, gelatinase A and B, and collagenase between His(16) and Ile(17). However, link N has been shown to act as a growth factor and stimulate synthesis of proteoglycans and collagen by chondrocytes of human articular cartilage. There are also no studies on the effect of link N on type IX collagen in any tissue. In the studies reported here, a serum-free pellet culture system has been used to examine whether link N can play a role in maintaining the integrity of disc matrix, specifically at the level of matrix assembly by cells of the IVD. Using this culture system, we determined the capacity of link N to stimulate accumulation of these matrix proteins in the annulus fibrosus (AF) and nucleus pulposus (NP). Gross inspection of separate AF and NP pellet cultures in the absence of link N revealed a progressive increase in size and a transition from "spherical" to "polygonal" pellets after centrifugation. Addition of 10 ng/ml link N resulted in increased pellet sizes for both AF and NP pellet cultures. Link N increased proteoglycan, type II and type IX collagen contents with an increase in DNA content over time. This study demonstrates that link N can act directly on disc cells to stimulate matrix production, which involves increased accumulation of proteoglycan, and types II and IX collagens. This study also identifies the value of pellet cultures for studies of the IVD cells in a serum-free chemically defined medium, in which pellets can continue growing in size in response to growth factors with minimal cell loss. Link N may have value in stimulating the growth and regeneration of the damaged IVD.

Effect of experimental parameters on the in vitro release kinetics of transforming growth factor beta1 from coral particles.

Coral bone graft substitutes have been supplemented in the past with growth factors to further enhance bone regeneration in defects. Little is known, however, on the dynamics of protein release from coral. Coral particles were studied for their ability to release transforming growth factor beta 1 (TGF-beta1) in vitro, under different adsorption conditions. Adsorption of TGF-beta1 (0.05 microg/mL) on coral particles (<80 microm or 300-450 microm) after 24 h of incubation was high, regardless of conditions. TGF-beta1 release kinetics in an artificial bone fluid followed a specific profile: an initial 1-h protein burst, followed by a decreasing release during the next 24 h at which time the release increased again to reach a constant rate until the end of the 2-week study. The protein release rate appeared mainly to depend upon the diffusion of TGF-beta1. TGF-beta1 release from coral particles was enhanced in the presence of bovine serum albumin (BSA) compared to the release in the presence of gelatin, and was dependent on the pH of adsorption. The highest total TGF-beta1 release was obtained when adsorption occurred with BSA at pH 7.4 (82 +/- 3%), while the lowest release was observed when adsorption was done in the presence of gelatin at pH 11 (38 +/- 1%). TGF-beta1 release was also found to vary with particle size, higher release being obtained with the smaller particles. These results suggest that coral particles could be used as a delivery system for growth factors, and that the release rate may be modulated through modification of the adsorption conditions and coral particle size.