Papers by Author: Racquel Z. LeGeros

Abstract: Bone is a composite of approximately 65% inorganic phase (carbonate apatite, CHA) and a 35% organic phase (mostly collagen). To date, several commercial composites consisting of natural or synthetic polymers and calcium phosphates ( hydroxyapatite, tricalcium phosphate, biphasic calcium phosphates) are recommended for use in bone repair. Objective: The aim of this study was to compare the physico-chemical properties of gelatin/carbonate apatite composites with that of bovine bone. Native (Gel) or cross-linked (Gel*) was used. Methods: The CHA was prepared by hydrolysis method. The gelatin (denatured collagen) was cross-linked using Genipin. The gelatin/CHA composite were prepared by mixing of 35% gelatin and 65% CHA and freeze-drying. The composites were characterized using x-ray diffraction (XRD), FT-IR spectroscopy, scanning electron microscopy (SEM) and thermogravimetry (TGA). Dissolution properties were determined in acidic buffer (0.1M KAc, pH 6, 37°C). Mechanical strength was determined using 3-point bend test. Bovine bone was similarly characterized for comparison. Results: The composition and crystallite size of the CHA were similar to that of the bone mineral. The Gel/CHA and Gel*/CHA composites showed several physico-chemical properties (crystallinity, composition, thermal stability, mechanical strength, dissolution rate) similar to that of bone. Gel*/CHA compared to Gel/CHA composites showed lower elastic modulus, flexural strength, dissolution rate, swelling and higher porosity. Conclusion: The Gel*/CHA composites presented several properties similar to those of bovine bone and may have potential as bone substitute materials.

Abstract: Ovariectomized rats were fed a diet containing minerals at high concentrations, such as Ca, P, and F (high-mineral diet), and changes in the femoral diaphysis were investigated after 24 weeks. The femur was mainly red and partially orange on the color scale of the 3D-map in Groups A and B, showing a high BMD. The region adjacent to the marrow cavity was yellow, showing a lower BMD than that in the outer region of the femur. In Group C, the red area was small in the outer region and the inner region was mainly yellow and green on the color scale. The inner region adjacent to the marrow cavity showed a view of unevenly resorbed bone, and the BMD was lower than those in Groups A and B. Incorporation of F into the body influences the apatite crystal structure and crystal growth, which subsequently influences adsorption of F to crystals and structural changes. Therefore, it is important to ingest F at the optimum concentration.

Abstract: Differences in structural and crystallinity between the lateral and medial regions of the central femoral diaphysis in low-mineral-diet-fed ovariectomized rats were investigated using polarization microscopy and Raman spectral analysis. Eighteen 19-week-old female Wistar rats were divided into 3 groups (Group 1: sham ovariectomy + normal diet group, Group 2: ovariectomy + normal diet group, Group 3: ovariectomy + low-mineral-diet group). Measurements were performed in the lateral and medial regions of the femoral sagittal cross-section at 24 weeks of feeding. On polarization microscopy, tubular structures of haversian and Volkmanns canals and osteocytes were observed in the femur in Groups 1 and 2. In Group 3, the number of these tubular structures in the femur was decreased compared to those in Groups 1 and 2. Moreover, the bone width of the femur decreased in Group 3 compared to those in Groups 1 and 2. On Raman spectral analysis, the peaks of organic and inorganic components were observed in the lateral and medial regions of the femur in all groups. In magnified views of the peak of ν₁PO₄^3-spectrum, the peaks in the lateral and medial regions overlapped in Group 1, but the peak of the medial region was about 0.3 and 0.8 cm^-1 shifted toward the low wavenumber side compared to those of the lateral region in Groups 2 and 3, respectively. Based on these findings, the influences of reduced estrogen secretion and the low-mineral diet were clarified as differences in the femoral structural and crystallinity on polarization microscopy and Raman spectral analysis. Since highly sensitive detailed point analysis can be performed within a short time using Raman spectral analysis, it may be useful for high-dimensional structural analysis.

Abstract: Natural bone constitutes of an inorganic phase (a biological nanoapatite) and an organic phase (mostly type I collagen). The challenge is to develop a material that can regenerate lost bone tissue with degradation and resorption kinetics compatible with the new bone formation. The aim of this study was to prepare self-organized magnesium and carbonate substituted apatite/collagen scaffolds, cross-linked with glutaraldehyde (GA). Bovine tendon was submitted to alkaline treatment resulting in a negatively charged collagen surface. The scaffolds were prepared by precipitation: simultaneous dropwise addition of solution containing calcium (Ca) and magnesium (Mg) ions and collagen into a buffered solution containing carbonate and phosphate ions in reaction vessel maintained at 37 °C, pH=8. The reaction products were cross-linked with 0.125 and 0.25% (v/v) glutaraldehyde (GA) solution and freeze-dried. The samples were characterized by Fourier-transformed infrared spectroscopy (FTIR). In vitro cytotoxicity (based on three parameters assays) and scaffolds degradation in culture medium and osteoblastic cells culture were performed in the cross-linked materials. No cytotoxic effects were observed. The cross-linked samples with the lower GA concentration showed a lower stability when placed in contact with culture medium. Human osteoblasts attached on the scaffolds surface cross-linked with 0.25% GA, forming a continuous layer after 14 days of incubation. These results showed potential application of the designed scaffolds for bone tissue engineering.

Abstract: Due to the lack of macroporosity in current available Calcium Phosphate cement used in osteoarticular surgery, Micro and Macroporous Biphasic CaP Cement (MCPC™) was developed. The MCPC™ concept was the association of a settable and a fast resorbable matrix and a sieved fraction of microporous biphasic calcium phosphate (BCP) granules, recognized for the high osteoconductive and osteogenic properties. During the resorption of the matrix, a porous structure is created and the osteoconductive effect of the granules promotes the bone ingrowth. A goat preclinical study was realized to evaluate the efficacy of MCPC™ for C3 and C4 vertebral body filling defects during 6 months. Bone remodelling was evidenced demonstrating bone ingrowth at the expense of the cement and surrounding the residual BCP granules. Bone trabeculae were observed coming from the spongious bone to the implant site. Human vertebral body filling cases demonstrated the biocompatibility and the safety of MCPC™ for bone reconstruction. Results of this study demonstrated the importance of special combination of calcium phosphate granules in the MCPC™ to provide macroporosity and scaffolding for newly formed bone.

Abstract: Both natural and synthetic materials have been utilized to provide three dimensional scaffold environments ideal for bone repair. The biomechanical and biocompatibility characteristics of these scaffolds play a vital role in successful tissue engineering constructs. Polymer/carbonate apatite (CHA) composites have shown to improve cell adhesion and proliferation on the scaffold as well as increase elastic modulus, toughness and strength. The aim of this study is to prepare CHA- polylactic-co-glycolide (PLGA) composites in the form of microsphere, scaffold and disc and evaluate their physico-chemical properties, mechanical properties and in vitro bioactivity. 3-D porous cylindrical composite scaffolds were prepared using PLGA/CHA composites with varying PLGA/CHA ratios (30:70 and 50:50). The CHA was prepared by hydrolysis method and characterized using x-ray diffraction (XRD) and Fourier Transform Infrared spectroscopy (FTIR). The physico-chemical and mechanical properties of the composite scaffolds were evaluated using scanning electron microscopy (SEM), micro-computed tomography (μCT), XRD, FTIR, and thermogravimetry (TGA). Flexural strength was determined using Instron. In vitro bioactivity was determined by the formation of apatite on composite disc surfaces after immersion in simulated body fluid (SBF). SEM and μCT analyses showed high porosity and interconnectivity between microspheres in the composite scaffolds. In vitro bioactivity was observed by the development of an apatite layer on the surfaces of the composite scaffolds after immersion in simulated body fluid. The mechanical strength of the scaffolds was to be dependent on the PLGA-CHA ratio. The elastic modulus, toughness and strength values obtained for the composites were similar to those of reported bone substituted materials. Results from this study provided information on the fabrication of PLGA-CHA scaffolds and their properties that may be useful for their potential application in bone repair and as scaffolds in tissue engineering for bone regeneration.

Abstract: Silk has been used in biomedical applications for centuries. The potential of silk for application in tissue engineering is currently being explored. The purpose of this study was to develop new method of mineralizing silk with carbonate apatite and determine cell response. The cocoons were placed in sodium bicarbonate solution and heat treated. The treated silk fibers were mineralized with carbonate apatite (CHA) using precipitation and new microwave methods. The mineralized and non-mineralized silks were characterized using SEM, EDS, XRD, FT-IR, and TGA. Cell response to mineralized silk and non-mineralized silk was determined using human osteoblast like cells (MG-63). The microwave method was more efficient than the precipitation method in terms of the amount of minerals incorporated with the silk and time required for mineralization. EDS, FT-IR and XRD identified CHA in the mineralized silk. In terms of cell response, greater production of type 1 collagen was observed with CHA mineralized silk compared with non-mineralized silk.

Abstract: The incorporation of magnesium in the synthetic apatite has been associated with biomineralization process and osteoporosis therapy in human and animals. Magnesium easily replaces calcium in the apatite lattice and influences or controls the hydroxyapatite crystallization processes. In this work, Mg-substituted calcium deficient apatite, with Mg/Ca ratio = 0.1, 0.15 and 0.2 were synthesized by precipitation method. Then, sintered at 1000 oC and compared with a commercial product labeled as tricalcium phosphate sintered at the 1000 oC. The sintered products showed tricalcium phosphate (β-TCP) structure. The Mg2+ substitution in the Ca(4) and Ca(5) sites of β-TCP and the lattice parameter changes were estimated using the Rietveld method. Using this method, the formulas Ca2.73(Mg0.27)(PO4)2, Ca2.71(Mg0.29)(PO4)2 and Ca2.70(Mg0.23Mg0.07)(PO4)2 were calculated for the samples with Mg/Ca ratio = 0.1, 0.15 and 0.2 respectively.