Email this article Current challenges in bone grafting revolve around the limited availability of autografts and the complications associated with their use. Promising alternatives include osteoinductive substances stimulating stem cells to mature into bone-forming osteoblasts. However, existing products lack optimal characteristics of a bone graft. The study aimed to evaluate the impact of L-arginine treatment on hydroxyapatite (HA) derived from ovine bone and compare its surface and mechanical properties to that of the commercially available xenograft-Bio-Oss.
The research was structured as an
A bone graft made of HA and L-arginine displayed a complex and interconnected pore structure, indicating that the sintering process had a significant impact. SEM confirmed this. FTIR analysis identified peaks at 650–700 cm−1 and 1000–1100 cm−1, confirming the presence of HA and L-arginine. X-ray Diffraction assessments also confirmed the existence of both substances in the sintered specimens, supporting their suitability for various biomedical applications.
The study presents a novel approach, deproteinizing a bone graft followed by sintering at 360°C with L-arginine. Physicochemical analyses confirmed desired mechanical attributes and surface characteristics. Further investigations are needed to evaluate cellular adherence, immunological response, and osteogenesis in relevant animal models.
Bones possess remarkable self-repairing capabilities, efficiently healing fractures or disruptions through natural processes. However, severe bone traumas resulting from trauma, neoplastic growths, any pathological or inherent malformations present significant therapeutic challenges beyond the reach of traditional interventions.[
Osteoinductive agents have a unique ability to stimulate the maturation of native stem cells into osteoblasts, the key cells responsible for bone tissue formation.[
This study aimed to assess the impact of L-arginine on hydroxyapatite (HA) from ovine bone and compare its properties with a commercial bone graft material (Bio-Oss). The main objective is to gather insights on the osteoinductive properties of bone graft materials and strive for innovative alternatives that match the effectiveness of autogenous bone grafts, recognized as the gold standard in bone restoration. Null hypothesis is framed as there is no difference between prepared novel bone graft with L-arginine and existing commercially available Bio-Oss in terms of physical and osteoconductive properties.
The present
Bone contains both organic and inorganic components. The organic part comprises of lipids and proteins, while the inorganic part includes compounds such as calcium carbonate, HA, calcium oxides, and phosphates. Hence, the first step in obtaining inorganic HA is to remove the organic components. The preparatory procedure involved the extraction of the organic constituents: segments of the ovine femur’s central region were sectioned into substantial slices, with any residual soft-tissue meticulously excised. Subsequently, to remove the intrinsic fluids, including marrow and soft tissues, the bone slices were subjected to a boiling regimen at 100°C for a duration spanning 12 h. Subsequent to the process of cleaning, the specimens were stored at a temperature of 4°C for an interval of 12 h, after which they were subjected to a thermal desiccation phase in a hot air oven set at 100°C, spanning another 12-h duration. Leftover soft tissue and unnecessary material from the samples were carefully removed. Subsequent chemical procedures aimed at the eradication of lipids, adipose content, and proteins through thermal extraction technique. Following a 12-h immersion phase, the specimens underwent a meticulous ethanol rinse to eliminate residual organic solvents. Subsequent to this ethanol cleansing, the samples were subjected to a thermal desiccation process in a hot air oven, maintained at 100°C, for an additional 12-h period. To thoroughly remove organic materials, 150 mL of Ethylene diamine (Cat. No: 70790, Sisco Research Laboratories Pvt. Ltd., Mumbai, India) was used as an extra solvent (HME 250, Labquest, Mumbai, India). This procedure was also conducted over 48-h duration. The specimens underwent dual ethanol washes [
Preparation of hydroxyapatite-based bone graft.
Scanning electron microscopy (SEM) serves as the modality for evaluating parameters such as particle dimensions, structural morphology, and surface characteristics. The samples were measured to ensure optimal placement and stability on the specimen stage subsequent to this, the granular entities underwent a sequential purification process involving acetone rinsing, deionization using distilled water, followed by air-drying phase to eliminate any residual moisture content. Utilizing a high-energy electron beam, SEM (Hitachi S-3400N, New Delhi, India) imagery of the specimen was procured. When electrons interacted with the sample, it caused the release of secondary electrons, electron back-scattering, and unique X-ray emissions. These signals were captured by detectors, and subsequently comprehensive visual representations were created. The surface intricacies of the samples were observed at 1 μm and 5 μm. JSM–IT800 NANO SEM apparatus (JEOL Benelux in the Netherlands), was used. Subsequent data analysis was done using the Image J software to ascertain the mean particle dimensions inferred from the SEM images.
X-ray diffraction (XRD) spectroscopy was used to study the mineralogical phases and the degree of crystalline structure in the samples from both the experimental and control groups. The finely ground samples were fixed onto the analytical holder, and the analysis was done using a D8 Advance Diffractometer (Bruker AXS GmbH, Karlsruhe, Germany), spanning a 2 θ angular range extending from 10° to 80°. X-ray emissions originated predominantly from a cathode ray tube, subsequently undergoing filtration processes to yield monochromatic radiation directed at the specimens. This resulted in generation of diffracted rays, which were captured, analyzed, and quantified. The X-ray wavelength was meticulously calibrated to approximate the bond distances inherent between atoms within the crystalline lattice, thereby producing a distinctive pattern reflective of the atomic configuration within the positioned samples. Subsequent analysis compared these findings with established benchmarks to ascertain the phase consistency and crystalline integrity. The resultant data sets were graphically represented using the origin software platform.
The sample was exposed to infrared radiation, and the resulting absorbance spectrum is obtained using a Fourier Transform Infrared Spectroscope (ALPHA II Compact Bruker, Billerica, Massachusetts). Key settings include spectral resolution and scanning range. The mechanism involves measuring the interferogram, from which the spectrum is derived, offering insights into molecular composition.
The samples were crushed and made into fine powder to assess for the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay. MTT assay was carried out to assess cell viability and proliferation (MTT cell growth assay kit, Sisco Research Laboratories, Mumbai, India). In the 6-well plate, 1 mL of complete culture medium per well was added. Next, 0.5 mg/mL MTT was added to the bottom well. The plate was then incubated at 37°C for 4 h. After incubation, the culture medium was aspirated from the insert well, and the resulting formazan crystals were eventually solubilized by adding 100 μL of dimethyl sulfoxide (DMSO) solution per well. Finally, 100 μL of the colored DMSO was transferred from each insert and each well to a new 96-well plate to quantify cell viability. Absorbance at 450 nm was measured using a microplate reader.
Percentage cell viability was calculated using the following equation:
Cell viability (%) = (A450 od of sample / A450 od of control) × 100
In a differentiation medium containing DMEMF12, 10 mM B-glycerophosphate, 0.05 mM ascorbic acid, and magnesium nanoparticles, the MG63 cells, osteoclast cells, were grown for 14 days using an osteogenesis kit (catalog N 5533-25G, Sigma-Aldrich, Burlington, United States). Calcium deposition was identified using alizarin red staining. The cells were once more stained with 2% alizarin red solution (catalog N 5533-25G, Sigma-Aldrich, Burlington, United States) for 10 min after 2 weeks. The cells were subsequently given two washes in 1X PBS. Each well received 200 μL of DMSO for quantitative analysis, which was incubated for 1 h. Using a spectrophotometer, the amount of alizarin was determined at 405 nm.
All experiments were performed in at least triplicates. The results are presented as mean ± standard deviation (SD) statistical analysis was done using SPSS software (Version 26.0; SPSS, Inc., Chicago, IL, USA). The errors of each data point were obtained from a minimum of three independent replicate tests, from which the mean and SDs were calculated. Comparative measurements between independent data sets were performed using one way ANOVA, significant level at 0.05.
HA-based bone graft samples, enhanced with L-Arginine, were examined alongside the commercial bone graft product BioOss. The analysis was done using SEM, XRD, Fourier-transform infrared spectroscopy (FTIR), and MTT assay.
The SEM examination of BioOss revealed a cohesive matrix characterized by closely interwoven particles with bioactive surface coating. The synthesized specimens exhibited a distinct pore arrangement coupled with a compact structure, showcasing a meticulously delineated surface topography when compared with BioOss. SEM assessments revealed a meticulously organized porosity, underscoring the synthesis of HA originating from ovine osseous material. In addition, the SEM images showcased a notably sleek surface texture accentuated by strong interparticle cohesion, further emphasizing the compact L-Arginine and HA matrix [
Scanning electron microscopy analysis. (a) Hydroxyapatite-based bone graft modified with L-arginine (Scale at 1 μm), (b) Hydroxyapatite-based bone graft (Scale at 5 μm), (c) BioOss (Scale at 1 μm).
Upon analysis of the XRD data, distinct crystalline phases of HA with the composition Ca4.854 (H0.126(PO4) 3) (OH) 0.834) and calcium oxide phosphate with the formula CA5(PO4) 3O0.5) were detected, accompanied by a presence of silica, L-arginine. Graphically, pronounced peaks of elevated intensity were seen at an approximate angle of 42.5o, 43.7o and 49.4o [
X-ray diffractometry analysis showing peaks of elevated intensity and confirming the presence of hydroxyapatite in commercially available bone graft-BioOss, ovine bone graft processed at 3600 C and Ovine bone graft + Arginine.
HA is identified by the presence of phosphate and carbonate groups. In FTIR analysis, peaks at 900–1200 cm−1 and 1400–1500 cm−1 confirm these groups, affirming the presence of HA. Samples showed bands in this range, confirming HA presence. An additional peak at 3200 cm−1 indicated L-arginine presence, suggesting its binding and dispersion in the HA matrix [
Fourier-transform infrared spectroscopy analysis (a) Hydroxyapatite-based bone graft modified with L-Arginine (b) BioOss showing peaks at 900–1200 cm−¹, 1400–1500 cm−¹ and 3200 cm−¹.
When considering the results of the MTT assay, all the samples showed biocompatibility and growth of MG-63 cells at 24 h [
Mean cell aggregation at 25 and 50 μg concentration of bone graft at 24 h.
Cell viability at 24 h showing cell aggregation (a) at 25 μg/mL concentration, (b) at 50 μg concentration, (c) control.
HA-based L-arginine-modified bone graft showed an increased amount of aggregation of the MG-63 cells after a period of 24 h at 25 and 50 μg/mL concentration of bone graft. This can greatly affect and also enhance the osteogenic differentiation potential [
Alizarin red staining (405 nm) for bone formation in dental pulp stem cells (25 μg/mL).
Within the confines of this research, the influence of L-arginine on HA sourced from ovine osseous material was meticulously explored. The synthesized HA specimens were compared with commercially available BioOss using advanced analytical modalities including SEM, XRD assessments, FTIR and MTT assay. The SEM examination of BioOss revealed a cohesive matrix with closely interwoven particles and bioactive surface coating. SEM assessments showed distinct pore arrangement and compact structure, with meticulously delineated surface topography. XRD analysis detected crystalline phases of HA and calcium oxide phosphate, aligning with BioOss peaks. FTIR confirmed HA presence with characteristic bands, and L-arginine binding indicated by a peak at 3200 cm−1. MTT assay indicated biocompatibility and MG-63 cell growth, with enhanced cell aggregation at 25 and 50 μg/mL concentrations of HA-based L-arginine-modified bone graft, potentially enhancing osteogenic differentiation potential. The results indicated similar characteristics as that of BioOss, supporting the potential use of L-arginine-modified HA bone graft in clinical scenarios after thorough and meticulous
Diverse studies have been carried out to establish various methodologies associated with the fabrication of HA derived from bovine bone. Notably, HA was synthesized from bovine bone utilizing a triphasic hydrothermal treatment approach.[
The influence of amino acids on bone grafts is a complex aspect studied in the field of biomaterials and tissue engineering. Incorporating L-arginine into bone graft material holds promising implications for cellular responses, tissue regeneration, and clinical success. Studies have indicated that L-arginine, as an essential amino acid, can positively influence osteoblast activity, enhance collagen synthesis, and promote angiogenesis.[
While our investigation provided valuable understanding regarding the influence of L-arginine on HA sourced from ovine bone, it is crucial to recognize a few inherent constraints. Primarily, our emphasis is directed toward the presence of potential variables such as the intrinsic chemicals and contaminants present in the initial stage of bone substrate processing. In addition, while our chosen analytical methods (including SEM, XRD, and FTIR assessments) offered insights into the microstructure, crystalline properties, and biological relevance of the sintered HA, there exists a scope for the exploration of even more sophisticated characterization methodologies. Emphasis on
Bovine bone xenografts available in the market undergo processing temperatures that span from 300°C to 1200°C. Elevated processing temperatures often result in enhanced crystallinity and a decelerated rate of bone resorption during the healing process. Subsequently, the bone graft with amino acid was assessed based on its physicochemical and biocompatible properties. From our observations, we postulate that bone grafts modified with L-arginine yielded a material suitable for grafting by demonstrating satisfactory mechanical attributes and surface features. However, future laboratory tests are essential to evaluate the attachment and growth of various osteogenic cell lines. This should be followed by detailed examinations to measure various additional factors such as immune response, graft breakdown, and the subsequent complications and their alternatives.
Nil.
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.