A Porous Fluoride-Substituted Bovine-Derived Hydroxyapatite Scaffold Constructed for Applications in Bone Tissue Regeneration.

Hydroxyapatite is widely used in bone implantation because of its similar mineral composition to natural bone, allowing it to serve as a biocompatible osteoconductive support. A bovine-derived hydroxyapatite (BHA) scaffold was developed through an array of defatting and deproteinization procedures. The BHA scaffold was substituted with fluoride ions using a modified sol-gel method to produce a bovine-derived fluorapatite (BFA) scaffold. Fourier-transform infrared spectroscopy and X-ray diffraction analysis showed that fluoride ions were successfully substituted into the BHA lattice. According to energy dispersive X-ray analysis, the main inorganic phases contained calcium and phosphorus with a fluoride ratio of ~1-2 wt%. Scanning electron microscopy presented a natural microporous architecture for the BFA scaffold with pore sizes ranging from ~200-600 µm. The BHA scaffold was chemically stable and showed sustained degradation in simulated-body fluid. Young's modulus and yield strength were superior in the BFA scaffold to BHA. In vitro cell culture studies showed that the BFA was biocompatible, supporting the proliferative growth of Saos-2 osteoblast cells and exhibiting osteoinductive features. This unique technique of producing hydroxyapatite from bovine bone with the intent of producing high performance biomedically targeted materials could be used to improve bone repair.

In vitro effects of wool-derived keratin on human dental pulp-derived stem cells for endodontic applications.

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration.

The role of dietary calcium in the etiology of childhood rickets in the past and the present.

For more than two centuries, lack of sunlight has been understood to cause vitamin D deficiency and documented as a primary cause of rickets. As such, evidence of rickets in the archeological record has been used as a proxy for vitamin D status in past individuals and populations. In the last decade, a clinical global consensus has emerged wherein it is recognized that dietary calcium deficiency also plays a role in the manifestation of rickets and classic skeletal deformities may not form if dietary calcium is normal even if vitamin D is deficient. This disease is now clinically called "nutritional rickets" to reflect the fact that rickets can take calcium deficiency-predominant or vitamin D deficiency-predominant forms. However, there are currently no paleopathological studies wherein dietary calcium deficiency is critically considered a primary etiology of the disease. We review here the interplay of calcium, vitamin D, and phosphorous in bone homeostasis, examine the role of dietary calcium in human health, and critically explore the clinical literature on calcium deficiency-predominant rickets. Finally, we report a case of rickets from the late Formative Period (~2500-1500 years ago) of the Atacama Desert and argue the disease in this infant is likely an example of calcium deficiency-predominant rickets. We conclude that most archeological cases of rickets are the result of multiple micronutrient deficiencies that compound to manifest in macroscopic skeletal lesions. For clinicians, these factors are important for implementing best treatment practice, and for paleopathologists they are necessary for appropriate interpretation of health in past communities.

The adaptive immune response to porous regenerated keratin as a bone graft substitute in an ovine model.

Reconstituted keratin is a novel bone graft material when prepared as a rigid scaffold. Understanding the immunogenicity of this material is important to determine whether this substance is a viable surgical option. Previous studies have shown no innate immune system activation in response to reconstituted keratin implants. To examine antibody-mediated immune responses to reconstituted keratin implants, bone and blood samples were taken from twelve sheep with surgically created tibial defects containing such implants. RT-PCR was used to detect mRNA of the inflammatory marker SOCS 3 in local bony tissue, and a novel immunohistochemistry assay developed to detect antikeratin antibodies in serum. Two animals were sacrificed per time-point at weeks 1, 2, 4, 6, 8 and 12. Time points for serum analysis included baseline (pre-surgery) and all other time points; mRNA analysis examined samples from all time points. No upregulation in antikeratin antibodies or SOCS 3 mRNA was observed at any time point, indicating that reconstituted keratin implants do not trigger an adaptive immune response in vivo in an ovine model. These findings provide the platform for further development of keratin implants in other mammalian models to define its immunogenic profile and safety.

Development and Characterization of a Biocomposite Material from Chitosan and New Zealand-Sourced Bovine-Derived Hydroxyapatite for Bone Regeneration.

A biocomposite scaffold was developed using chitosan (CS) and bovine-derived hydroxyapatite (BHA). The prepared CS-BHA biocomposite scaffold was characterized for its physiochemical and biological properties and compared against control BHA scaffolds to evaluate the effects of CS. Energy-dispersive X-ray analysis confirmed the elemental composition of the CS-BHA scaffold, which presented peaks for C and O from CS and Ca and P along with trace elements in the bovine bone such as Na, Mg, and Cl. Fourier transform infrared spectroscopy confirmed the presence of phosphate, hydroxyl, carbonate, and amide functional groups attributed to the CS and BHA present in the biocomposite scaffolds. The CS-BHA scaffolds demonstrated an interconnected porous structure with pore sizes ranging from 60 to 600 µm and a total porosity of ∼64-75%, as revealed by scanning electron microscopy and micro-CT analyses, respectively. Furthermore, thermogravimetric analysis revealed that the CS-BHA scaffold lost 70% of its weight when heated up to 1000 °C, which is characteristic of CS phase decomposition in the biocomposite. In vitro studies demonstrated that the CS-BHA scaffolds were biocompatible toward Saos-2 osteoblast-like cells, showing high cell viability and a significant increase in cell proliferation across the measured timepoints compared to the controls.

Effect of chitosan infiltration on hydroxyapatite scaffolds derived from New Zealand bovine cancellous bones for bone regeneration.

Hydroxyapatite (HA) derived from bovine bones garnered wider interest as a bone substitute due to their abundant availability as meat wastes and similarities in morphology and mineral composition to human bone. In our previous work, we developed an easy and reproducible method to prepare xenograft HA scaffolds from NZ bovine cancellous bones (BHA). However, the processing methodology rendered the material mechanically weak. The present study investigated the infiltration of chitosan (CS) into the bovine HA scaffolds (CSHA) to improve the mechanical properties of BHA. The presence of characteristic functional groups of HA and CS as detected by infrared spectroscopy confirmed the infiltration of CS into the BHA scaffolds. X-ray Diffraction study confirmed the presence of the hydroxyapatite phase in both BHA and CSHA scaffolds. SEM and µCT analyses showed the CSHA scaffolds presented adequate porosity and an interconnected porous architecture required for cell migration and attachment. CSHA scaffolds presented good thermal, chemical and structural stability while demonstrating sustained biodegradability in simulated body fluid. CSHA scaffolds presented mechanical properties significantly higher than the BHA scaffolds. CSHA scaffolds were biocompatible with Saos-2 osteoblast cells and supported cell proliferation significantly better than the BHA scaffolds indicating their potential in bone tissue engineering.

Hydroxyapatite-polymer biocomposites for bone regeneration: A review of current trends.

Bone tissue engineering has emerged as one of the most indispensable approaches to address bone trauma in the past few decades. This approach offers an efficient and a risk-free alternative to autografts and allografts by employing a combination of biomaterials and cells to promote bone regeneration. Hydroxyapatite (HA) is a ceramic biomaterial that mimics the mineral composition of bones and teeth in vertebrates. HA, commonly produced via several synthetic routes over the years has been found to exhibit good bioactivity, biocompatibility, and osteoconductivity under both in vitro and in vivo conditions. However, the brittle nature of HA restricts its usage for load bearing applications. To address this problem, HA has been used in combination with several polymers in the form of biocomposite implants to primarily improve its mechanical properties and also enhance the implants' overall performance by simultaneously exploiting the positive effects of both HA and the polymer involved in making the biocomposite. This review article summarizes the past and recent developments in the evolution of HA-polymer biocomposite implants as an "ideal" biomaterial scaffold for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2046-2057, 2018.