Phosphate Graft Material Research Articles

A new multiphase calcium phosphate graft material improves bone healing-An in vitro and in vivo analysis.

This study aims to evaluate the potential of a novel biomaterial synthesized from amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), and hydroxyapatite (HA) to repair critical-sized defects (CSD) in rabbit calvaria. In vitro analyses of cell viability, cell proliferation, formation of mineral nodules, and cell differentiation using qPCR were performed for comparing experimental calcium phosphate (ECP), deproteinized bovine bone (DBB), and beta-tricalcium phosphate (β-TCP). Bilateral CSDs were created in 45 rabbit calvaria. Six groups were evaluated: ECP, ECP + fibrin sealant (ECP + S), coagulum, autogenous bone, DBB, and β-TCP. Euthanasia was performed at 2, 4, and 8 weeks, followed by micro-computed tomography and histological and immunohistochemical analyses. Results from in vitro analyses revealed similar biocompatibility for all tested materials and a tendency for higher gene expression of some bone markers in the ECP group than in β-TCP and DBB groups at 7 days. In contrast to that in DBB and β-TCP groups, ECP displayed growing bone volume over total volume percentage (BV/TV%) with time in vivo. Histological analysis revealed a greater number of giant cells and reduced size of grafted particles in ECP during all periods of analysis. RUNX-2 expression was statistically lower in ECP than DBB at 2 and 4 weeks. Despite no statistical significance, ECP presented the highest absolute values for ALP-expression at 2, 4, and 8 weeks compared with other groups. Together, our findings indicate that a combination of the ACP, OCP, and HA phases into ECP is beneficial and promising for bone regeneration.

The addition of a polyglutamate domain to the angiogenic QK peptide improves peptide coupling to bone graft materials leading to enhanced endothelial cell activation.

Vascularization of bone grafts is vital for graft integration and bone repair, however non-autologous graft sources have limited potential to induce angiogenesis. Accordingly, intensive research has focused on functionalizing non-autologous materials with angiogenic factors. In the current study we evaluated a method for coupling an angiogenic peptide to the surface of two clinically-relevant graft materials, anorganic bovine bone (ABB) and synthetic hydroxyapatite (HA). Specifically, the VEGF-derived “QK” peptide was synthesized with a heptaglutamate (E7) domain, a motif that has strong affinity for calcium phosphate graft materials. Compared with unmodified QK, a 4–6 fold enrichment was observed in the binding of E7-modified QK (E7-QK) to ABB and HA. The E7-QK peptide was then assessed for its capacity to stimulate angiogenic cell behaviors. Human umbilical vein endothelial cells (HUVECs) were treated with solutions of either QK or E7-QK, and it was found that QK and E7-QK elicited equivalent levels of cell migration, tubule formation and activation of the Akt and ERK signaling pathways. These data confirmed that the inherent bioactivity of the QK sequence was not diminished by the addition of the E7 domain. We further verified that the activity of E7-QK was retained following peptide binding to the graft surface. HA disks were coated with QK or E7-QK, and then HUVECs were seeded onto the disks. Consistent with the increased amount of E7-QK bound to HA, relative to QK, markedly greater activation of Akt and ERK 1/2 was observed in cells exposed to the E7-QK-coated disks. Taken together, these results suggest that the E7 domain can be leveraged to concentrate angiogenic peptides on graft materials, facilitating delivery of higher peptide concentrations within the graft site. The ability to endow diverse graft materials with angiogenic potential holds promise for augmenting the regenerative capacity of non-autologous bone grafts.

Comparative Study on the Application of Mesenchymal Stromal Cells Combined with Tricalcium Phosphate Scaffold into Femoral Bone Defects

This prospective study sought to evaluate the healing quality of implanted ultraporous β-tricalcium phosphate sown with expanded autologous mesenchymal stromal cells (MSCs) into femoral defects during revision hip arthroplasty. A total of 37 osseous defects in 37 patients were treated and evaluated concerning bone regeneration. Nineteen subjects received β-tricalcium phosphate graft material serving as a carrier of expanded autologous MSCs (the trial group A), nine subjects received β-tricalcium phosphate graft material only (the study group B) and nine subjects received cancellous allografts only (the control group C). Clinical and radiographic evaluations were scheduled at 6 weeks, 3, 6, and 12 months post-operatively, and performed at the most recent visit as well. All observed complications were recorded during follow-up to assess the use of an ultraporous β-tricalcium phosphate synthetic graft material combined with expanded MSCs in bone defect repair. The resulting data from participants with accomplished follow-up were processed and statistically evaluated with a Freeman–Halton modification of the Fischer’s exact test, a P < 0.05 value was considered to be significant. Whereas no significant difference was observed between the trial group A with β-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs and control group C with cancellous impaction allografting in terms of the bone defect healing, significant differences were documented between the study group B with β-tricalcium phosphate graft material only and control group C. Regarding adverse effects, six serious events were recorded during the clinical trial with no causal relationship to the cell product. β-tricalcium phosphate synthetic graft material serving as a carrier of expanded autologous MSCs appears safe and promotes the healing of bone defects in a jeopardized and/or impaired microenvironment. This clinical trial was registered at the EU Clinical Trials Register before patient recruitment (Registration number: EudraCT number 2012-005599-33; Date of registration: 2013-02-04).

Osteostimulatory effect of bone grafts on fibroblast cultures.

Objective:We analyzed the morphological changes and alkaline phosphatase (ALP) level in fibroblast, which is indicative of their functional ability when cultured in three different commercially available graft materials with osseoconductive property.Materials and Methods:Fibroblasts obtained from fifth passage were seeded within three different bone substitutes (bovine hydroxyapatite [HA] [Osseo-graft®], β-tricalciumphosphate [RTR®], bovine HA [Bio-oss®]) and incubated under standard cell culture conditions. 10 samples in each group were evaluated for cell morphology and alkaline phosphates activity using scanning electron microscopy and spectrophotometric analysis on the 7th day of culture.Results:Fibroblast cultured with RTR® showed changes in morphology and increase in ALP activity when compared to fibroblast cultured with Osseo-graft® and Bio-oss®.Conclusion:Alkaline phosphatase activity was observed in fibroblasts when cultured with three types of commercially available bone grafts. ALP activity was highest when cultured with β-tricalcium phosphate graft material indicating its better bone regenerating capacity of this graft material.

Comparison of osteoconductive properties of three different β-tricalcium phosphate graft materials: A pilot histomorphometric study in a pig model

AimsThe aim of this study was to compare the de novo bone formation ability and osteoconductive effects of three different β-tricalcium phosphate (β-TCP) graft materials. The micro-architectural parameters of the newly formed bone tissues were also compared among the different graft materials. Material and methodsEight male Swiss domestic pigs were used in the study. Five bony defects were made with a trephine bur. Three of the defects were filled with Cerasorb®, Kasios® and Poresorb®. The fourth defect was filled with an autogenous bone graft. The last defect remained empty. All subjects were sacrificed after 8 weeks. ResultsWhen compared to a negative control group, significant healing was observed in all the groups except the Cerasorb group. The osteoconductivity of the Poresorb group was better than that of the other groups (p < 0.05). The difference in the osteoconductivity of the Kasios and Cerasorb groups was statistically significant (p < 0.05). Comparison of the micro-architectural properties of newly formed bone tissues retrieved from the defects showed that those filled with Poresorb were the best. Conclusionβ-TCP materials show different results in terms of the volume and characteristics of new bone formation, although they have a similar chemical structure.

Effect of a tunnel-structured β-tricalcium phosphate graft material on periodontal regeneration: a pilot study in a canine one-wall intrabony defect model.

Tissue regeneration is affected by the porosity, chemical properties and geometric structure of graft materials. Regeneration of severe periodontal defects, such as one-wall intrabony defects, is difficult because of reduced tissue support, and bone grafts are commonly used in such cases. In the present study, a tunnel-structured β-tricalcium phosphate (tunnel β-TCP) graft material designed to stimulate bone formation was fabricated. The objective of this pilot study was to evaluate the effect of this graft material on periodontal regeneration in one-wall intrabony defects in dogs. Six male beagle dogs were used in this study. First, the mandibular second and third incisors were extracted. Experimental surgery was performed 12 wk after tooth extraction. Bilateral 4 × 8 mm (width × depth) one-wall intrabony defects were created in the mesial side of the mandibular canines. At the experimental sites, the defects were filled with tunnel β-TCP, whereas the control defects were left empty. Twelve weeks after surgery, qualitative and quantitative histological analyses were performed. There were no signs of clinical inflammation 12 wk after surgery. Coronal extension indicative of new bone formation was higher at the experimental sites than at the control sites, although the differences between both the sites in the newly formed cementum and connective tissue attachment were not significant. Newly formed periodontal ligament and cementum-like tissue were evident along the root surface at the experimental sites. The inner surface of the tunnels was partially resorbed and replaced with new bone. New blood vessels were observed inside the lumens of tunnel β-TCP. Tunnel β-TCP serves as a scaffold for new bone formation in one-wall intrabony defects.

Effect of Silicon-Doped Calcium Phosphate Bone Substitutes on Bone Formation and Osteoblastic Phenotype Expression &lt;i&gt;In Vivo&lt;/i&gt;

This study evaluates the effect of two novel particulate silicon-doped calcium phosphate graft materials as compared to the currently clinically used material β-TCP on osteogenesis and bone formation after implantation in critical-size defects the sheep scapula. These materials were developed in order to create biodegradable bone substitute materials that degrade rapidly, but still stimulate osteogenesis at the same time, thereby resulting in bone repair and regeneration with fully functional bone tissue. All bone substitute materials studied facilitated excellent bony regeneration of critical-size defects in the sheep scapula. Of the three grafting materials studied, the calcium alkali orthophosphate material with the crystalline phase Ca2KNa (PO4)2, with a small amorphous portion containing magnesium potassium phosphate and a small addition of sodium magnesium silicate had the greatest stimulatory effect on bone formation and expression of osteogenic markers, while exhibiting the highest biodegradability.

Effects of Laser and Ozone Therapies on Bone Healing in the Calvarial Defects

This study aims to analyze the effect of the low-level laser therapy (LLLT) and ozone therapy on the bone healing of critical size defect (CSD) in rat calvaria. A total of 30 Wistar male rats were used. A 5-mm-diameter trephine bur was used to create CSD on the right side of the parietal bone of each rat calvarium. Once the bone was excised, a synthetic biphasic calcium phosphate graft material was implanted to all the bone defect sites. The animals were randomly divided into 3 groups as follows: the control group (n = 10), which received no LLLT or ozone therapy; the LLLT group (n = 10), which received only LLLT (120 seconds, 3 times a week for 2 weeks); and the ozone therapy group (n = 10) (120 seconds, 3 times a week for 2 weeks). After 1 month, all the rats were killed, and the sections were examined to evaluate the presence of inflammatory infiltrate, connective tissue, and new bone formation areas. Histomorphometric analyses showed that in the LLLT and ozone groups, the new bone areas were significantly higher than in the control group (P < 0.05). In the LLLT group, higher new bone areas were found than in the ozone group (P < 0.05). This study demonstrated that both ozone and laser therapies had a positive effect on bone formation in rat calvarial defect, compared with the control group; however, ozone therapy was more effective than LLLT (808 nm; 0.1 W; 4 J/cm(2); 0.028 cm(2), continuous wave mode).

Allogeneic mesenchymal progenitor cells for posterolateral lumbar spine fusion in sheep

Background contextOsteoconductive porous ceramic bone graft materials supplemented with mesenchymal precursor cells (MPC) derived from autologous bone marrow aspirates have been shown to stimulate successful interbody and posterolateral spine fusion in preclinical models. Recent advances in immunomagnetic cell sorting have enabled purification and isolation of pluripotent stem cells from marrow aspirates and have expanded stem cell technology to allogeneic cell sources. Allogeneic MPC technology combined with appropriate synthetic biomaterial carriers could provide both the osteogenic and osteoconductive components needed for successful posterolateral spine fusion without the need for autologous bone harvest or expensive recombinant protein technology. PurposeTo determine the safety and efficacy of a hydroxyapatite:tricalcium phosphate graft material supplemented with allogeneic mesenchymal precursor cells in posterolateral lumbar spine fusion using an ovine model. Study designSkeletally mature ewes underwent single-level instrumented posterolateral lumbar spine fusion using either autograft (AG), hydroxyapatite:tricalcium phosphate carrier (CP), or CP supplemented with allogeneic mesenchymal progenitor cells (MPCs). Three doses of MPCs were evaluated: 25 × 106 cells (low dose, LD), 75 × 106 cells (mid dose, MD), and 225 × 106 cell (high dose, HD). Animals survived for either 4 or 9 months. MethodsPlain radiographs were acquired and scored for bridging bone at regular intervals during healing to monitor fusion development. Hematology, coagulation, and serum chemistry were monitored at regular intervals throughout the study to monitor animal health. After necropsy, computed tomography, high-resolution radiography, biomechanical testing, organ pathology, bone histopathology, and bone histomorphometry were conducted to monitor the safety and ascertain the efficacy of MPC treatment. ResultsMPC treatment in this spine fusion model resulted in no observed adverse systemic or local tissue responses. Radiographically, fusion scores for MPC-treated animals were uniformly higher compared with those treated with carrier alone (CP) after 3 months and continued the same trend throughout 9 month of healing. Quantitative computed tomography confirmed better connectivity of the fusion for MPC treatment groups compared with CP. Biomechanical analyses were not able to differentiate between treatment groups. Histomorphometry results confirmed radiographic and quantitative computed tomography results; cell-supplemented treatment groups and autograft had equivalent amounts of bone within the fusion mass and less bony fusion tissue was found within the fusion mass in specimens from the CP treatment group. No conclusive effects of cell dose of fusion efficacy were noted. ConclusionsAdult allogeneic mesenchymal precursor cells delivered via a hydroxyapatite:tricalcium phosphate carrier were both safe and efficacious in this ovine spine fusion model. Results from this preclinical study support that allogeneic mesenchymal precursor cells produced fusion efficacy similar to that achieved using iliac crest autograft, thereby providing a safe and viable option to achieve successful posterolateral spine fusion.

Bone healing response to a synthetic calcium sulfate/β‐tricalcium phosphate graft material in a sheep vertebral body defect model

The introduction of a material able to promote osteogenesis and remodelling activity in a clinically relevant time frame in vertebroplasty and kyphoplasty procedures may have patient benefit. We report the in-vivo performance of a biphasic synthetic bone graft material (Genex Paste, Biocomposites, UK) [test material], composed of calcium sulfate and β-tricalcium phosphate, implanted into a sheep vertebral defect model. Cavities drilled into 4 adjacent vertebrae (L2 to L5) of 24 skeletally mature sheep were; (1) filled with the test material; (2) filled with commercially available polymethylmethacrylate [PMMA] cement; (3) remained empty [sham]. Analysis was performed immediately after implantation and at 8, 16, and 36 weeks post implantation. Sites were evaluated for bone growth with microCT analysis, histological examination, and mechanical testing under compression. The test material exhibited an improved tissue response over the PMMA, indicating a superior biological tolerance. MicroCT and histology indicated marked osteoregenerative capacity of the test material when compared with sham and the PMMA. The percentage of new bone formation was higher for the test material than sham at 16 and 36 weeks post implantation, with bone regeneration almost complete at 36 weeks in this group. Resorption of test material and the integration into new bone tissue were demonstrated. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans

The aim of this study was to examine the efficacy of a new biphasic hydroxyapatite/tricalcium phosphate (HA/TCP) bone substitute in combination with particulate autogenous bone in sinus floor augmentation procedures. A simultaneous or a two-stage sinus augmentation and implant placement were conducted in 28 patients. A mixture of HA/TCP and autogenous bone chips in a 1 : 1 ratio was used as the grafting biomaterial. Cylindrical specimen bone retrieval was performed in all patients except one. Specimens were harvested either at 6 (n=14) or 9 (n=13) months post-augmentation. For histologic and histomorphometric evaluations, the non-decalcified tissue processing (Donath's technique) was performed. Newly formed bone around the grafted particles was found in all samples. The encircling, highly cellular bone followed the outline of the grafted particles in direct contact. Both woven and lamellar types of bone were observed. Morphometrically, the total mean bone area fraction of all sections was 34.8+/-10.3%, increasing from 28.6+/-7.8% at 6 months to 41.6+/-8.3% at 9 months (P<0.001). Mean particle area fraction average was 25.5+/-11.6% and 23.5+/-9.3% at 6 and 9 months, respectively, with a total mean of 24.5+/-10.4%. The increase in bone area fraction was not significantly correlated to the decrease of the grafted particles area fraction. The biphasic HA/TCP showed biocompatible and osteoconductive properties. This alloplast as a composite with autogenous bone chips promotes newly formed bone, which increases in its fraction along an extended healing period.

Histomorphometric assessment of bone formation in sinus augmentation utilizing a combination of autogenous and hydroxyapatite/biphasic tricalcium phosphate graft materials: at 6 and 9 months in humans

AbstractObjective: The aim of this study was to examine the efficacy of a new biphasic hydroxyapatite/tricalcium phosphate (HA/TCP) bone substitute in combination with particulate autogenous bone in sinus floor augmentation procedures.Material and methods: A simultaneous or a two‐stage sinus augmentation and implant placement were conducted in 28 patients. A mixture of HA/TCP and autogenous bone chips in a 1 : 1 ratio was used as the grafting biomaterial. Cylindrical specimen bone retrieval was performed in all patients except one. Specimens were harvested either at 6 (n=14) or 9 (n=13) months post‐augmentation. For histologic and histomorphometric evaluations, the non‐decalcified tissue processing (Donath's technique) was performed.Results: Newly formed bone around the grafted particles was found in all samples. The encircling, highly cellular bone followed the outline of the grafted particles in direct contact. Both woven and lamellar types of bone were observed.Morphometrically, the total mean bone area fraction of all sections was 34.8±10.3%, increasing from 28.6±7.8% at 6 months to 41.6±8.3% at 9 months (P&lt;0.001). Mean particle area fraction average was 25.5±11.6% and 23.5±9.3% at 6 and 9 months, respectively, with a total mean of 24.5±10.4%. The increase in bone area fraction was not significantly correlated to the decrease of the grafted particles area fraction.Conclusions: The biphasic HA/TCP showed biocompatible and osteoconductive properties. This alloplast as a composite with autogenous bone chips promotes newly formed bone, which increases in its fraction along an extended healing period.