ABSTRACT
Though crucial for natural bone healing, local calcium ion (Ca2+) and phosphate ion (Pi) concentrations can exceed the cytotoxic limit leading to mitochondrial overload, oxidative stress, and cell death. For bone tissue engineering applications, H2S can be employed as a cytoprotective molecule to enhance mesenchymal stem cell (MSC) tolerance to cytotoxic Ca2+/Pi concentrations. Varied concentrations of sodium hydrogen sulfide (NaSH), a fast-releasing H2S donor, were applied to assess the influence of H2S on MSC proliferation. The results suggested a toxicity limit of 4 mM for NaSH and that 1 mM of NaSH could improve cell proliferation and differentiation in the presence of cytotoxic levels of Ca2+ (32 mM) and/or Pi (16 mM). To controllably deliver H2S over time, a novel donor molecule (thioglutamic acid-GluSH) was synthesized and evaluated for its H2S release profile. Excitingly, GluSH successfully maintained cytoprotective level of H2S over 7 days. Furthermore, MSCs exposed to cytotoxic Ca2+/Pi concentrations in the presence of GluSH were able to thrive and differentiate into osteoblasts. These findings suggest that the incorporation of a sustained H2S donor such as GluSH into CaP-based bone graft substitutes can facilitate considerable cytoprotection, making it an attractive option for complex bone regenerative engineering applications.
ABSTRACT
Vertebral compression fractures account for approximately 700,000 out of the 1.5 million total osteoporotic fractures that occur annually in the USA. There is growing interest in substituting currently utilized clinical treatments for vertebral compression fractures with an injectable, degradable, and bioactive system. In this research we studied the osteoinductive effect of calcium phosphate incorporation into cellulose nanocrystal/chitosan hydrogels with varying ratios of carbonate as an ionic crosslinker and genipin as a covalent crosslinker. As calcium and phosphate ions have been shown to be osteoinductive in time and concentration dependent manners, dibasic calcium phosphate was chosen as a bioactive additive due to its desirable controlled ion delivery potential. Gelation time, swelling ratio, erosion, compressive strength, and ion release behavior of different dibasic calcium phosphate incorporated hydrogels were evaluated. Mesenchymal stem cells were then exposed to mechanically competent hydrogels found capable of maintaining calcium and phosphate concentrations within the established bioactive range in order to assess their cytotoxicity and osteoinductivity. Our results demonstrate that hydrogels with higher covalent crosslinking possessed better mechanical properties and stabilities as well as more controlled calcium and phosphate ion release. Interestingly, dibasic calcium phosphate incorporation not only improved hydrogel bioactivity but also resulted in greater compressive strength.
Subject(s)
Biocompatible Materials/chemistry , Calcium Phosphates/administration & dosage , Drug Carriers/chemistry , Fractures, Compression/therapy , Spinal Fractures/therapy , Animals , Cell Line , Chitosan/chemistry , Drug Compounding/methods , Humans , Hydrogels/chemistry , Materials Testing , Mesenchymal Stem Cells/drug effects , Mice , Nanoparticles/chemistry , Osteogenesis/drug effectsABSTRACT
Vertebral compression fractures are a very common consequence of osteoporosis for which injection of a non-biodegradable, non-bioactive, mechanically-stiff polymer bone cement into the vertebral body is the most common treatment. Recently, there has been growing interest in using bioactive, degradable, and bone biomechanics-matching products as an alternative approach for treating these fractures. In this research, we focused on creating injectable, chitosan-based hydrogels that can convey mechanical strength similar to vertebral bone as well as possess inherent osteoinductivity. First, we investigated the effects of three different factors - 1) bioactive phosphate ionic crosslinking; 2) genipin covalent crosslinking; 3) mechanically reinforcing cellulose nanocrystal incorporation - on the material properties of chitosan-based hydrogels. Mesenchymal stem cells were then exposed to hydrogels with optimum mechanical properties and stability in order to assess the biological effects of the bioactive phosphate ionic crosslinker. Our results show that hydrogels with higher ionic and covalent crosslinking ratios supplemented with neutral cellulose nanocrystals possessed desirable compressive strength and stability. Also, the significant osteoinductivity of these composite hydrogels demonstrated their potential to function as an injectable system for the future treatment of vertebral compression fractures.
