Chondrogenesis of Mesenchymal Stem Cells through Local Release of TGF-ß3 from Heparinized Collagen Biofabric.

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high-density pellets of MSCs is generally attained by soluble exogenous TGF-ß3 in culture media, which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the postimplantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro. In this vein, a heparin-conjugated mechanically robust collagen fabric was developed for sustained delivery of TGF-ß3. The amount of conjugated heparin was varied to enhance the amount of TGF-ß3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-ß3 for up to 8 days of culture, which resulted in 15-fold increase in GAG production, and six-fold increase in collagen synthesis with respect to the No TGF-ß3 group. The resulting matrix was cartilage like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-ß3 administration resulted in a Young's modulus of ∼600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration. Impact statement The most significant finding of this study was that sustained release of TGF-ß3 from heparinized collagen scaffold had chondroinductive effect on pelleted human mesenchymal stem cells (hMSCs). The effect was comparable to that observed in hMSC pellets that were cultured in chondrogenic media supplemented with TGF-ß3. The stiffness of scaffolds at the baseline was about 50% that of native cartilage and over 28 days the combined stiffness of pellet/scaffold complex converged to the stiffness of native cartilage. These data indicate that the scaffold system can generate a load-bearing cartilage-like tissue by using hMSCs pellets in a mechanically competent framework.

Heparin-mediated antibiotic delivery from an electrochemically-aligned collagen sheet.

BACKGROUND: Implantable medical devices and hardware are prolific in medicine, but hardware associated infections remain a major issue. OBJECTIVE: To develop and evaluate a novel, biologic antimicrobial coating for medical implants. METHODS: Electrochemically compacted collagen sheets with and without crosslinked heparin were synthesized per a protocol developed by our group. Sheets were incubated in antibiotic solution (gentamicin or moxifloxacin) overnight, and in vitro activity was assessed with five-day diffusion assays against Pseudomonas aeruginosa. Antibiotic release over time from gentamicin-infused sheets was determined using in vitro elution and high performance liquid chromatography (HPLC). RESULTS: Collagen-heparin-antibiotic sheets demonstrated larger growth inhibition zones against P. aeruginosa compared to collagen-antibiotic alone sheets. This activity persisted for five days and was not impacted by rinsing sheets prior to evaluation. Rinsed collagen-antibiotic sheets did not produce any inhibition zones. Elution of gentamicin from collagen-heparin-gentamicin sheets was gradual and remained above the minimal inhibitory concentration for gentamicin-sensitive organisms for 29 days. Conversely, collagen-gentamicin sheets eluted their antibiotic load within 24 hours. Overall, heparin-associated sheets demonstrated larger inhibition zones against P. aeruginosa and prolonged elution profile via HPLC. CONCLUSION: We developed a novel, local antibiotic delivery system that could be used to coat medical implants/hardware in the future and reduce post-operative infections.

Diffuse microdamage in bone activates anabolic response by osteoblasts via involvement of voltage-gated calcium channels.

INTRODUCTION: Matrix damage sustained by bone tissue is repaired by the concerted action of bone cells. Previous studies have reported extracellular calcium ([Ca2+]E) efflux to originate from regions of bone undergoing diffuse microdamage termed as "diffuse microdamage-induced calcium efflux" (DMICE). DMICE has also been shown to activate and increase intracellular calcium ([Ca2+]I) signaling in osteoblasts via the involvement of voltage-gated calcium channels (VGCC). Past studies have assessed early stage (< 1 h) responses of osteoblasts to DMICE. The current study tested the hypothesis that DMICE has longer-term sustained effect such that it induces anabolic response of osteoblasts. MATERIALS AND METHODS: Osteoblasts derived from mouse calvariae were seeded on devitalized bovine bone wafers. Localized diffuse damage was induced in the vicinity of cells by bending. The response of osteoblasts to DMICE was evaluated by testing gene expression, protein synthesis and mineralized nodule formation. RESULTS: Cells on damaged bone wafers showed a significant increase in RUNX2 and Osterix expression compared to non-loaded control. Also, RUNX2 and Osterix expression were suppressed significantly when the cells were treated with bepridil, a non-selective VGCC inhibitor, prior to loading. Significantly higher amounts of osteocalcin and mineralized nodules were synthesized by osteoblasts on diffuse damaged bone wafers, while bepridil treatment resulted in a significant decrease in osteocalcin production and mineralized nodule formation. CONCLUSION: In conclusion, this study demonstrated that DMICE activates anabolic responses of osteoblasts through activation of VGCC. Future studies of osteoblast response to DMICE in vivo will help to clarify how bone cells repair diffuse microdamage.

Repetitive short-span application of extracellular calcium is osteopromotive to osteoprogenitor cells.

Extracellular calcium ([Ca2+ ]E ) concentration has been suggested to stimulate osteoblastic activity; thus, calcium can be used to enhance fracture healing. However, systemic administration of calcium at high dose levels may cause physiological problems such as hypercalcaemia. Short-span application of single or repetitive [Ca2+ ]E stimulus may be suggested as a novel regimen to reduce such side effects. However, osteopromotive effect of such short-term [Ca2+ ]E stimulus on osteoprogenitor cells has not yet been evaluated yet. This study investigated the effects of [Ca2+ ]E dose (6 and 18 mM) and regimen (single, repetitive, and continuous) on viability, proliferation, osteogenic gene expression, and mineral formation by osteoprogenitor cells. BMP-2 treatment was set as the positive control group. It was observed that repetitive and continuous calcium stimulation resulted in significant enhancement of osteoblastic activity. A 6 mM [Ca2+ ]E significantly increased cell viability and proliferation in all three regimens, and the expression of osteogenic transcription factors was significantly upregulated by continuous application of 6 mM [Ca2+ ]E . It was observed that application of [Ca2+ ]E repetitively at 18 mM had an osteopromotive effect to an extent that was as pronounced as BMP-2. Continuous application of 18 mM [Ca2+ ]E provided the greatest degree of osteogenic activity among all groups. This study demonstrated that repetitive [Ca2+ ]E exposure from the basal aspect of cells resulted in upregulation of osteogenic transcription factor and bone formation. The knowledge gained from the dose and treatment regimen of calcium therapy is important in setting the guidelines for developing approaches to treat fractures.

Activation of intracellular calcium signaling in osteoblasts colocalizes with the formation of post-yield diffuse microdamage in bone matrix.

Previous studies demonstrated that extracellular calcium efflux ([Ca(2+)]E) originates from the regions of bone extracellular matrix that are undergoing microdamage. Such [Ca(2+)]E is reported to induce the activation of intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells. The current study investigated the association between microdamage and local activation of intracellular calcium signaling quantifiably in MC3T3-E1 cells. Cells were seeded on devitalized notched bovine bone samples to induce damage controllably within the field of observation. A sequential staining procedure was implemented to stain for intracellular calcium activation followed by staining for microdamage on the same sample. The increase in [Ca(2+)]I fluorescence in cells of mechanically loaded samples was greater than that of unloaded negative control cells. The results showed that more than 80% of the cells with increased [Ca(2+)]I fluorescence were located within the damage zone. In conclusion, the findings demonstrate that there are spatial proximity between diffuse microdamage induction and the activation of intracellular calcium ([Ca(2+)]I) signaling in MC3T3-E1 cells. The downstream responses to the observed activation in future research may help understand how bone cells repair microdamage.

Novel Raman Spectroscopic Biomarkers Indicate That Postyield Damage Denatures Bone's Collagen.

Raman spectroscopy has become a powerful tool in the assessment of bone quality. However, the use of Raman spectroscopy to assess collagen quality in bone is less established than mineral quality. Because postyield mechanical properties of bone are mostly determined by collagen rather than the mineral phase, it is essential to identify new spectroscopic biomarkers that help infer the status of collagen quality. Amide I and amide III bands are uniquely useful for collagen conformational analysis. Thus, the first aim of this work was to identify the regions of amide bands that are sensitive to thermally induced denaturation. Collagen sheets and bone were thermally denatured to identify spectral measures that change significantly following denaturation. The second aim was to assess whether mechanical damage denatures the collagen phase of bone, as reflected by the molecular spectroscopic biomarkers identified in the first aim. The third aim was to assess the correlation between these new spectroscopic biomarkers and postyield mechanical properties of cortical bone. Our results revealed five peaks whose intensities were sensitive to thermal and mechanical denaturation: ∼1245, ∼1270, and ∼1320 cm(-1) in the amide III band, and ∼1640 and ∼1670 cm(-1) in the amide I band. Four peak intensity ratios derived from these peaks were found to be sensitive to denaturation: 1670/1640, 1320/1454, 1245/1270, and 1245/1454. Among these four spectral biomarkers, only 1670/1640 displayed significant correlation with all postyield mechanical properties. The overall results showed that these peak intensity ratios can be used as novel spectroscopic biomarkers to assess collagen quality and integrity. The changes in these ratios with denaturation may reflect alterations in the collagen secondary structure, specifically a transition from ordered to less-ordered structure. The overall results clearly demonstrate that this new spectral information, specifically the ratio of 1670/1640, can be used to understand the involvement of collagen quality in the fragility of bone. © 2015 American Society for Bone and Mineral Research.

Microdamage induced calcium efflux from bone matrix activates intracellular calcium signaling in osteoblasts via L-type and T-type voltage-gated calcium channels.

Mechanisms by which bone microdamage triggers repair response are not completely understood. It has been shown that calcium efflux ([Ca(2+)]E) occurs from regions of bone undergoing microdamage. Such efflux has also been shown to trigger intracellular calcium signaling ([Ca(2+)]I) in MC3T3-E1 cells local to damaged regions. Voltage-gated calcium channels (VGCCs) are implicated in the entry of [Ca(2+)]E to the cytoplasm. We investigated the involvement of VGCC in the extracellular calcium induced intracellular calcium response (ECIICR). MC3T3-E1 cells were subjected to one dimensional calcium efflux from their basal aspect which results in an increase in [Ca(2+)]I. This increase was concomitant with membrane depolarization and it was significantly reduced in the presence of Bepridil, a non-selective VGCC inhibitor. To identify specific type(s) of VGCC in ECIICR, the cells were treated with selective inhibitors for different types of VGCC. Significant changes in the peak intensity and the number of [Ca(2+)]I oscillations were observed when L-type and T-type specific VGCC inhibitors (Verapamil and NNC55-0396, respectively) were used. So as to confirm the involvement of L- and T-type VGCC in the context of microdamage, cells were seeded on devitalized notched bone specimen, which were loaded to induce microdamage in the presence and absence of Verapamil and NNC55-0396. The results showed significant decrease in [Ca(2+)]I activity of cells in the microdamaged regions of bone when L- and T-type blockers were applied. This study demonstrated that extracellular calcium increase in association with damage depolarizes the cell membrane and the calcium ions enter the cell cytoplasm by L- and T-type VGCCs.