Extraction of Hydroxyapatite from Camel Bone for Bone Tissue Engineering Application.

Waste tissues such as mammalian bone are a valuable source from which to extract hydroxyapatite. Camel bone-based hydroxyapatite (CBHA) was extracted from the femur of camel bones using a defatting and deproteinization procedure. The extracted CBHA was mechanically, chemically, physically, morphologically and structurally characterized. Fourier-Transform Infra-Red (FTIR) spectra, Micro-Raman, and X-ray diffraction analysis confirmed successful extraction of hydroxyapatite. The mechanical properties of the CBHA scaffold were measured using a Universal Instron compression tester. Scanning electron microscopy showed the presence of a characteristic interconnected porous architecture with pore diameter ranging from 50-600 µm and micro-computer tomography (Micro-CT) analysis identified a mean porosity of 73.93. Thermogravimetric analysis showed that the CBHA was stable up to 1000 °C and lost only 1.435% of its weight. Inductively coupled plasma-mass spectrometry (ICP-MS) and Energy-dispersive-X-ray (EDX) analysis demonstrated the presence of significant amounts of calcium and phosphorus and trace ions of sodium, magnesium, zinc, lead and strontium. Following 21 days of incubation in simulated body fluid (SBF), the pH fluctuated between 10-10.45 and a gradual increase in weight loss was observed. In conclusion, the extracted CBHA is a promising material for future use in bone tissue regeneration applications.

Morphological alterations of the cornea following crosslinking treatment (CXL).

INTRODUCTION: Corneal crosslinking (CXL) has revolutionized the treatment of keratoconus during the past decade. In the present study, the morphological changes in the corneal collagen fibrils (CFs) following crosslinking treatment are described. MATERIALS AND METHODS: Ten pairs of porcine and rabbit corneas were retrieved. In each pair, one cornea was the control and the other underwent CXL treatment. The central corneal thickness (CCT) was measured before and after CXL treatment. Each treated and control cornea was examined with light microscopy and by transmission electron microscopy. RESULTS: (a) The mean CCT was significantly reduced following treatment. (b) CFs were more closely packed in the anterior region and loosely packed in the posterior region. (c) CF diameter increased significantly in the anterior and intermediate regions but declined gradually towards the deeper regions. (d) There was a statistically significant decrease in the interfibrillar distance over the different regions of the cornea, except for the posterior region in porcine corneas, where there was no change. (e) The distance between adjacent collagen lamellae was significantly decreased in all regions of treated rabbit corneas. There was no change in porcine corneas. CONCLUSION: CXL treatment resulted in increased the CF diameter and decreased interfibrillar distance in the anterior and intermediate regions, while its effects on the posterior region differed among species. The effect on interlamellar distance was more prominent in the rabbit model than the porcine model. CXL treatment stiffened the corneas by increasing CF diameter and decreasing interfibrillar distance in both rabbit and pig corneas.

Lyophilised Platelet-Rich Fibrin: Physical and Biological Characterisation.

BACKGROUND: Platelet-rich fibrin (PRF) has gained popularity in craniofacial surgery, as it provides an excellent reservoir of autologous growth factors (GFs) that are essential for bone regeneration. However, the low elastic modulus, short-term clinical application, poor storage potential and limitations in emergency therapy use restrict its more widespread clinical application. This study fabricates lyophilised PRF (Ly-PRF), evaluates its physical and biological properties, and explores its application for craniofacial tissue engineering purposes. MATERIAL AND METHODS: A lyophilisation method was applied, and the outcome was evaluated and compared with traditionally prepared PRF. We investigated how lyophilisation affected PRF's physical characteristics and biological properties by determining: (1) the physical and morphological architecture of Ly-PRF using SEM, and (2) the kinetic release of PDGF-AB using ELISA. RESULTS: Ly-PRF exhibited a dense and homogeneous interconnected 3D fibrin network. Moreover, clusters of morphologically consistent cells of platelets and leukocytes were apparent within Ly-PRF, along with evidence of PDGF-AB release in accordance with previously reports. CONCLUSIONS: The protocol established in this study for Ly-PRF preparation demonstrated versatility, and provides a biomaterial with growth factor release for potential use as a craniofacial bioscaffold.

Potential of Lyophilized Platelet Concentrates for Craniofacial Tissue Regenerative Therapies.

OBJECTIVE: The use of platelet concentrates (PCs) in oral and maxillofacial surgery, periodontology, and craniofacial surgery has been reported. While PCs provide a rich reservoir of autologous bioactive growth factors for tissue regeneration, their drawbacks include lack of utility for long-term application, low elastic modulus and strength, and limited storage capability. These issues restrict their broader application. This review focuses on the lyophilization of PCs (LPCs) and how this processing approach affects their biological and mechanical properties for application as a bioactive scaffold for craniofacial tissue regeneration. MATERIALS AND METHODS: A comprehensive search of five electronic databases, including Medline, PubMed, EMBASE, Web of Science, and Scopus, was conducted from 1946 until 2019 using a combination of search terms relating to this topic. RESULTS: Ten manuscripts were identified as being relevant. The use of LPCs was mostly studied in in vitro and in vivo craniofacial bone regeneration models. Notably, one clinical study reported the utility of LPCs for guided bone regeneration prior to dental implant placement. CONCLUSIONS: Lyophilization can enhance the inherent characteristics of PCs and extends shelf-life, enable their use in emergency surgery, and improve storage and transportation capabilities. In light of this, further preclinical studies and clinical trials are required, as LPCs offer a potential approach for clinical application in craniofacial tissue regeneration.

ST2 regulates bone loss in a site-dependent and estrogen-dependent manner.

Interleukin-33 (IL-33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2-deficient mice (ST2-/- ) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary-intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real-time polymerase chain reaction (qPCR). Deletion of IL-33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL-33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2-/- OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin-10 expression was decreased in both WT OVX and ST2-/- mice, and this reduction was completely restored in ST2-/- OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy-induced bone loss is IL-33/ST2-dependent in the maxilla but not in the femur, indicating a bimodal and site-specific role of ST2 in bone remodeling.

Current perspectives on corneal collagen crosslinking (CXL).

Corneal collagen crosslinking has revolutionized the treatment of keratoconus and post-refractive corneal ectasia in the past decade. Corneal crosslinking with riboflavin and ultraviolet A is proposed to halt the progression of keratectasia. In the original "Conventional Dresden Protocol" (C-CXL), the epithelium is removed prior to the crosslinking process to facilitate better absorption of riboflavin into the corneal stroma. Studies analyzing its short- and long-term outcomes revealed that although there are inconsistencies as to the effectiveness of this technique, the advantages prevail over the disadvantages. Therefore, corneal crosslinking (CXL) is widely used in current practice to treat keratoconus. In an attempt to improve the visual and topographical outcomes of C-CXL and to minimize time-related discomfort and endothelial-related side effects, various modifications such as accelerated crosslinking and transepithelial crosslinking methods have been introduced. The comparison of outcomes of these modified techniques with C-CXL has also returned contradictory results. Hence, it is difficult to clearly identify an optimal procedure that can overcome issues associated with the CXL. This review provides an up-to-date analysis on clinical and laboratory findings of these popular crosslinking protocols used in the treatment of keratoconus. It is evident from this review that in general, these modified techniques have succeeded in minimizing the immediate complications of the C-CXL technique. However, there were contradictory viewpoints regarding their effectiveness when compared with the conventional technique. Therefore, these modified techniques need to be further investigated to arrive at an optimal treatment option for keratoconus.

Monetite and brushite coated magnesium: in vivo and in vitro models for degradation analysis.

The use of magnesium (Mg) as a biodegradable metallic replacement of permanent orthopaedic materials is a current topic of interest and investigation. The appropriate biocompatibility, elastic modulus and mechanical properties of Mg recommend its suitability for bone fracture fixation. However, the degradation rates of Mg can be rapid and unpredictable resulting in mass hydrogen production and potential loss of mechanical integrity. Thus the application of calcium phosphate coatings has been considered as a means of improving the degradation properties of Mg. Brushite and monetite are utilized and their degradation properties (alongside uncoated Mg controls) are assessed in an in vivo subcutaneous environment and the findings compared to their in vitro degradation behaviour in immersion tests. The current findings suggest monetite coatings have significant degradation protective effects compared to brushite coatings in vivo. Furthermore, it is postulated that an in vitro immersion test may be used as a tentative predictor of in vivo subcutaneous degradation behavior of calcium phosphate coated and uncoated Mg.

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 calibration and in vivo validation of phenomenological corrosion models for resorbable magnesium-based orthopaedic implants.

Metallic bioresorbable orthopaedic implants based on magnesium, iron and zinc-based alloys that provide rigid internal fixation without foreign-body complications associated with permanent implants have great potential as next-generation orthopaedic devices. Magnesium (Mg) based alloys exhibit excellent biocompatibility. However, the mechanical performance of such implants for orthopaedic applications is contingent on limiting the rate of corrosion in vivo throughout the bone healing process. Additionally, the surgical procedure for the implantation of internal bone fixation devices may impart plastic deformation to the device, potentially altering the corrosion rate of the device. The primary objective of this study was to develop a computer-based model for predicting the in vivo corrosion behaviour of implants manufactured from a Mg-1Zn-0.25Ca ternary alloy (ZX10). The proposed corrosion model was calibrated with an extensive range of mechanical and in vitro corrosion testing. Finally, the model was validated by comparing the in vivo corrosion performance of the implants during preliminary animal testing with the corrosion performance predicted by the model. The proposed model accurately predicts the in vitro corrosion rate, while overestimating the in vivo corrosion rate of ZX10 implants. Overall, the model provides a "first-line of design" for the development of new bioresorbable Mg-based orthopaedic devices. STATEMENT OF SIGNIFICANCE: Biodegradable metallic orthopaedic implant devices have emerged as a potential alternative to permanent implants, although successful adoption is contingent on achieving an acceptable degradation profile. A reliable computational method for accurately estimating the rate of biodegradation in vivo would greatly accelerate the development of resorbable orthopaedic implants by highlighting the potential risk of premature implant failure at an early stage of the device development. Phenomenological corrosion modelling approach is a promising computational tool for predicting the biodegradation of implants. However, the validity of the models for predicting the in vivo biodegradation of Mg alloys is yet to be determined. Present study investigates the validity of the phenomenological modelling approach for simulating the biodegradation of resorbable metallic orthopaedic implants by using a porcine model that targets craniofacial applications.

Development, physicochemical characterization and in-vitro biocompatibility study of dromedary camel dentine derived hydroxyapatite for bone repair.

This study aimed to produce hydroxyapatite from the dentine portion of camel teeth using a defatting and deproteinizing procedure and characterize its physicochemical and biocompatibility properties. Biowaste such as waste camel teeth is a valuable source of hydroxyapatite, the main inorganic constituent of human bone and teeth which is frequently used as bone grafts in the biomedical field. Fourier Transform infrared (FTIR), and micro-Raman spectroscopy confirmed the functional groups as-sociated with hydroxyapatite. X-ray diffraction (XRD) studies showed camel dentine-derived hydroxyapatite (CDHA) corresponded with hydroxyapatite spectra. Scanning electron micros-copy (SEM) demonstrated the presence of dentinal tubules measuring from 1.69-2.91 µm. The inorganic phases of CDHA were primarily constituted of calcium and phosphorus, with trace levels of sodium, magnesium, potassium, and strontium, according to energy dispersive X-ray analysis (EDX) and inductively coupled plasma mass spectrometry (ICP-MS). After 28 days of incubation in simulated body fluid (SBF), the pH of the CDHA scaffold elevated to 9.2. in-vitro biocompatibility studies showed that the CDHA enabled Saos-2 cells to proliferate and express the bone marker osteonectin after 14 days of culture. For applications such as bone augmentation and filling bone gaps, CDHA offers a promising material. However, to evaluate the clinical feasibility of the CDHA, further in-vivo studies are required.

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.

Surgically-induced deformation in biodegradable orthopaedic implant devices.

The biocompatibility and mechanical performance of biodegradable metals (e.g. magnesium, iron, and zinc-based alloys) in orthopaedic-targeted applications are contingent on limiting the rate of corrosion in vivo throughout the bone healing. Concurrently, the surgical procedure for the implantation of internal bone fixation devices may impart plastic deformation to the device, potentially altering the corrosion rate of the device. However, the potential effect of the surgical implantation procedure on the mechanochemical performance of metallic degradable orthopaedic devices in vivo remains largely unresolved. The objective of the present study is to develop a robust technique that permits the quantification of the strain introduced due to surgical implantation of degradable orthopaedic devices. Specifically, a novel combined experimental-modelling approach based on 3D laser scanning in situ and the finite element method is utilised to quantify the plastic strain introduced to a bone fixation plate following surgical implantation in a cadaveric porcine model where the plate is based on a ternary magnesium-zinc-calcium alloy (ZX10). The magnitude of plastic strains determined by the above approach for the Mg craniofacial miniplate confirms that the surgical procedure itself has the potential to enhance the corrosion rate of the Mg alloy in an accelerated and potentially localised manner. STATEMENT OF SIGNIFICANCE: Biodegradable metallic orthopaedic implant devices have emerged as a potential alternative to permanent implants, although successful adoption is contingent on achieving an acceptable degradation profile. Plastic strain that is introduced to the device during surgical implantation may influence the resulting degradation behaviour of the implant. In the present work, 3D laser scanning is combined with computer simulation to estimate the level and distribution of surgically-induced plastic strain in a magnesium alloy (ZX10). Subsequently, clinically-relevant pre-strain is shown to influence the rate of corrosion of ZX10 in vitro, indicating the value of such an approach in the design of biodegradable metallic devices under multiaxial loading.

A novel classification of bone graft materials.

Over the past few decades, the field of biomaterials concerning bone tissue engineering has gained a significant amount of interest. This has led to new biomaterials to be used as bone substitute materials. Despite the rapid increase in the types and forms of bone substitutes, a comprehensive classification encompassing all types of bone graft materials that have so far been developed and evaluated is lacking. Therefore, this review aims to integrate and bring together the published data on bone substitutes within the last 5 years and produce a novel classification that would provide bone material researchers with a better understanding of what materials have so far been used and evaluated and the areas, where research is lacking and deserves more attention. The literature available in all major databases was obtained and filtered using an elimination criterion to extract all the articles related to studies that tested bone substitute materials in nonclinical and clinical trials over the last 5 years. The review article would provide bone material researchers with an insight into the materials that have been evaluated for bone tissue engineering applications and identify future perspectives for bone graft material research.

Wool keratin - A novel dietary protein source: Nutritional value and toxicological assessment.

Keratin derived protein (KDP) was extracted from sheep wool using high pressure microwave technology and food acids and investigated for its potential as a novel dietary protein. The proximate composition, amino acid profile, element profile, in vitro cytotoxicity and digestibility of KDP were evaluated. Nutritive effects of KDP at 50% dietary supplementation were compared with a casein-based diet in a growing rat model for 95 days. Results indicate KDP to be rich in protein (86%), amino acid cysteine (8.8 g/100 g) and element selenium (0.29 µg/g). KDP was non-cytotoxic in vitro at ≤ 2 mg/mL concentration. There were no differences in the rat's weight gain compared to the control group (P > 0.05). Overall, the inclusion of the KDP in the diet was an effective substitute for casein protein at 50% and KDP has the potential to be used in the food industry as a novel dietary protein, free of fat and carbohydrate.

Influence of food consistency on growth and morphology of the mandibular condyle.

The objective of this study was to determine if variation in the shape and mineralization of the mandibular condyle are the result of natural adaptation in response to different functional loading demands. Eight female Kuni Kuni piglets were randomly assigned to two groups of four, receiving either a soft or hard diet. Each animal was given three separate doses of vital stains intravenously at set time points during the study. At 8.5 months, animals were euthanized and temporomandibular joints (TMJs) were excised. Histological analysis was used to measure the amount of new bone deposition in the anterior, central, and posterior regions of the mandibular condyle. Backscatter electron (BSE) imaging was used as a semiquantitative estimate of bone mineralization in these two diet groups. Histology revealed that the degree of new bone deposition in the hard-diet group was significantly (n = 4, P < 0.001, paired t-test) higher than that of the soft-diet group. Also, the majority (87%) of animals fed a hard diet tended to show greater new bone deposition on the leftside in comparison to the right, indicating a chewing preference for the left side. In both groups, the degree of new bone deposition was significantly (P < 0.01) higher in the posterior area than in other regions. BSE imaging corroborated basic histology results, with significantly (P < 0.01) higher mineralization levels detected in the hard-diet group. These findings indicate that diet consistency has a small but significant effect on the rate of bone deposition in the mandibular condyle.

Animal model with structural similarity to human corneal collagen fibrillar arrangement.

Rabbit and porcine corneas have been used in scientific research due to their structural similarity to the human cornea. Currently, there are no studies that have compared corneal collagen fibrillar diameter, interfibrillar distance and interlamellar distance between human and animal models. Ten pairs of porcine, rabbit, and human corneas were used. These were analysed using light and Transmission Electron microscopy. The collagen fibrillar diameter, interfibrillar distance and interlamellar distance were statistically compared between porcine, rabbit and human corneas. The human, porcine and rabbit; mean collagen fibrillar diameters were: 24.52 ± 2.09 nm; 32.87 ± 0.87 nm; and 33.67 ± 1.97 nm. The mean interfibrillar distances were: 46.10 ± 2.44 nm; 53.33 ± 2.24 nm; and 52.87 ± 2.73 nm, respectively. The collagen fibrillar diameter and interfibrillar distance of porcine and rabbit corneas were significantly different (p < 0.001) to the human corneal values but not form each other. The interlamellar distance of human, porcine and rabbit corneas was: 2190 ± 820 nm; 6460 ± 1180 nm; and 4410 ± 1330 nm, respectively. All the comparisons were statistically different, in porcine versus rabbit at the p < 0.01 level and both porcine and rabbit versus human at the p < 0.001 level. Histologically, all five layers (epithelium, Bowman's layer, stroma, Descemet membrane and endothelium) of the cornea were visible in all the three species. While neither animal model was structurally identical to the human cornea, they are both relatively close to being used as models to study the biomechanical effects of external insults/treatments to be extrapolated to the human cornea.

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.

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.

Effect of cross-linking on microstructure and physical performance of casein protein.

The development of advanced materials from biorenewable protein biopolymers requires the generation of more exogenous bonds to maintain the microstructure and durability in the final products. Casein is the main protein of milk, representing about 80% of the total protein. In the present investigation the casein protein was solubilized and/or emulsified in aqueous alkaline solutions, and 2D films and 3D matrices were produced. The effects of silane (3-aminopropyl triethoxy silane), DL-glyceraldehyde and glutaraldehyde on tensile properties and water swelling/absorption of 2D casein films and also the microstructure of the freeze-dried 3D matrices were analyzed. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that there were no significant changes in the molecular weight (19-23.9 kDa) of the casein proteins on exposure to alkaline solutions of sodium hydroxide and silane. The casein films produced without glycerol plasticizer and with heat treatment (130 °C for 18 h) were fragile. However, the fragile films were transformed into ductile and tough materials on exposure to moisture (i.e., conditioned for one week at 50 ± 2% relative humidity and 22 ± 2 °C) and showed a maximum average tensile strength of 49-52 MPa and modulus of 1107-1391 MPa. The chemical cross-linkers (i.e., DL-glyceraldehyde and glutaraldehyde) improved the microstructure of glycerol plasticized casein protein, when analyzed under scanning electron microscope (SEM). Furthermore, these chemical cross-linking agents enhanced the mechanical properties and water resistant properties of casein films.