Tetracalcium phosphate and biphasic tetracalcium phosphate/tricalcium phosphate powders' effects evaluation on human osteoblasts.

BACKGROUND: Calcium ions levels in bone niches have been demonstrated to severely influence new bone formation. Osteoinductive scaffolds containing calcium have been largely studied to control the release of calcium in bone regeneration and tissue engineering purpose. The aim of the present study was, firstly, to synthesize two different resorbable calcium phosphate-based powders, thought to be reservoirs of calcium ions, and secondary, to investigate their effects on human osteoblasts, in order to develop a suitable titanium coating material. METHODS: Tetracalcium phosphate (A450) and biphasic tetracalcium phosphatae/tricalcium phosphate (A850) powders were prepared with an innovative method. The presence of calcium phosphate structures was chemically confirmed with XRD. Furthermore, powders macroscopic aspect was observed with a stereomicroscope. For in-vitro experiments, human osteoblastic cells were cultured in the presence of A450 and A850, and assayed for viability and metabolic activity through Crystal Violet and MTT, respectively. RESULTS: Our synthesis led to the formation of calcium phosphates in both samples, even though A850 presented a higher level of crystallinity and a more powdery aspects than A450. Both the samples enhanced the viability of cultured cells, inhibiting cell metabolic activity in the case of A850, which furthermore showed to be internalized by cells. CONCLUSIONS: We developed two different kind of calcium phosphate-based powders and we tested their effect on human osteoblasts, underlying the possibility of use calcium phosphate-based coatings to enhance cell response on implantable materials.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro

Abstract Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. Objective: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. Material and Methods: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. Results: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. Conclusions: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Stanozolol promotes osteogenic gene expression and apposition of bone mineral in vitro.

Stanozolol (ST) is a synthetic androgen with high anabolic potential. Although it is known that androgens play a positive role in bone metabolism, ST action on bone cells has not been sufficiently tested to support its clinical use for bone augmentation procedures. OBJECTIVE: This study aimed to assess the effects of ST on osteogenic activity and gene expression in SaOS-2 cells. MATERIAL AND METHODS: SaOS-2 deposition of mineralizing matrix in response to increasing doses of ST (0-1000 nM) was evaluated through Alizarin Red S and Calcein Green staining techniques at 6, 12 and 24 days. Gene expression of runt-related transcription factor 2 (RUNX2), vitamin D receptor (VDR), osteopontin (SPP1) and osteonectin (ON) was analyzed by RT-PCR. RESULTS: ST significantly influenced SaOS-2 osteogenic activity: stainings showed the presence of rounded calcified nodules, which increased both in number and in size over time and depending on ST dose. RT-PCR highlighted ST modulation of genes related to osteogenic differentiation. CONCLUSIONS: This study provided encouraging results, showing ST promoted the osteogenic commitment of SaOS-2 cells. Further studies are required to validate these data in primary osteoblasts and to investigate ST molecular pathway of action.

Tailoring the Interface of Biomaterials to Design Effective Scaffolds.

Tissue engineering (TE) is a multidisciplinary science, which including principles from material science, biology and medicine aims to develop biological substitutes to restore damaged tissues and organs. A major challenge in TE is the choice of suitable biomaterial to fabricate a scaffold that mimics native extracellular matrix guiding resident stem cells to regenerate the functional tissue. Ideally, the biomaterial should be tailored in order that the final scaffold would be (i) biodegradable to be gradually replaced by regenerating new tissue, (ii) mechanically similar to the tissue to regenerate, (iii) porous to allow cell growth as nutrient, oxygen and waste transport and (iv) bioactive to promote cell adhesion and differentiation. With this perspective, this review discusses the options and challenges facing biomaterial selection when a scaffold has to be designed. We highlight the possibilities in the final mold the materials should assume and the most effective techniques for its fabrication depending on the target tissue, including the alternatives to ameliorate its bioactivity. Furthermore, particular attention has been given to the influence that all these aspects have on resident cells considering the frontiers of materiobiology. In addition, a focus on chitosan as a versatile biomaterial for TE scaffold fabrication has been done, highlighting its latest advances in the literature on bone, skin, cartilage and cornea TE.

"Over-inlay" block graft and differential morphometry: a novel block graft model to study bone regeneration and host-to-graft interfaces in rats.

PURPOSE: The aim of this study was to present new a model that allows the study of the bone healing process, with an emphasis on the biological behavior of different graft-to-host interfaces. A standardized "over-inlay" surgical technique combined with a differential histomorphometric analysis is presented in order to optimize the use of critical-size calvarial defects in pre-clinical testing. METHODS: Critical-size defects were created into the parietal bone of 8 male Wistar rats. Deproteinized bovine bone (DBBM) blocks were inserted into the defects, so that part of the block was included within the calvarial thickness and part exceeded the calvarial height (an "over-inlay" graft). All animals were sacrificed at 1 or 3 months. Histomorphometric and immunohistochemical evaluation was carried out within distinct regions of interest (ROIs): the areas adjacent to the native bone (BA), the periosteal area (PA) and the central area (CA). RESULTS: The animals healed without complications. Differential morphometry allowed the examination of the tissue composition within distinct regions: the BA presented consistent amounts of new bone formation (NB), which increased over time (24.53%±1.26% at 1 month; 37.73%±0.39% at 3 months), thus suggesting that this area makes a substantial contribution toward NB. The PA was mainly composed of fibrous tissue (71.16%±8.06% and 78.30%±2.67%, respectively), while the CA showed high amounts of DBBM at both time points (78.30%±2.67% and 74.68%±1.07%, respectively), demonstrating a slow remodeling process. Blood vessels revealed a progressive migration from the interface with native bone toward the central area of the graft. Osterix-positive cells observed at 1 month within the PA suggested that the periosteum was a source of osteoprogenitor elements. Alkaline phosphatase data on matrix deposition confirmed this observation. CONCLUSIONS: The present model allowed for a standardized investigation of distinct graft-to-host interfaces both at vertically augmented and inlay-augmented sites, thus possibly limiting the number of animals required for pre-clinical investigations.

Role of prostaglandin E2 in the modulation of Wnt canonical signaling in cells on microstructured titanium surfaces.

BACKGROUND: Rough surface topography enhances the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of the modulation of prostaglandin E2 (PGE2) signaling on osteoblastic differentiation on titanium surfaces for endosseous implants with different topographies. METHODS: C2C12 cells were plated on polished or acid-etched/sand-blasted (SLA) titanium discs and stimulated with 1 µM PGE2 or 100 nM cyclooxygenase inhibitor indomethacin. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by real-time polymerase chain reaction (RT-PCR). Osteoblastic MC3T3 cells were then plated on polished or SLA titanium discs with or without indomethacin, and their proliferation and the expression of osteoblast-specific genes was assessed by RT-PCR. Cell morphology was furthermore studied on SEM, and cell adhesion was assessed by fluorescent labeling of focal adhesion. RESULTS: PGE2 decreased Wnt signaling stimulation in cells growing on polished or SLA surfaces, while indomethacin increased the expression of Wnt target genes in C2C12 and MC3T3 cells, by reporter assay. Moreover, indomethacin increased the expression of early differentiation marker alkaline phosphatase in MC3T3 cells on polished discs and of late marker osteocalcin in cells on SLA titanium. CONCLUSIONS: Prostaglandin signaling affects the activation of Wnt canonical pathway in osteoblastic and mesenchymal cells on microstructured surfaces.

The effect of age, gender, and insertion site on marginal bone loss around endosseous implants: results from a 3-year trial with premium implant system.

OBJECTIVES: The goal of this study was to evaluate bone changes around endosseous implants in partially edentulous patients. MATERIALS AND METHODS: A total of 632 two-stage implants were placed in 252 patients. The implants had straight emergence profile, ZirTi surface, 3.3 to 5 mm diameter, and 8.5 to 13 mm length. Bone levels were assessed on orthopantomography immediately after surgery and after 36 months and marginal bone loss (MBL) was calculated from their difference. RESULTS: Cumulative survival rate was 98.73%. Overall MBL was 0.8 mm ± 0.03 (mean ± SEM). Higher MBL was observed around implants in the maxilla than in the mandible (P < 0.007). A relation between implant diameter and MBL (P < 0.0001) was observed in male and, more limitedly, female patients. Older patients had higher MBL in the maxilla, but not in the mandible (P < 0.0001). MBL progressively increased with age in male patients, but reached a peak already in the 50-60 years age group in the female subset (P < 0.001). CONCLUSIONS: The overall MBL is consistent with the available literature. Site difference and patient age and gender appear to significantly affect MBL, representing important factors to be considered during implant placement.

Correlation between density and resorption of fresh-frozen and autogenous bone grafts.

TRIAL DESIGN: This analysis compared the outcome of fresh-frozen versus autologous bone block grafts for horizontal ridge augmentation in patients with Cawood and Howell class IV atrophies. METHODS: Seventeen patients received autologous grafts and 21 patients received fresh-frozen bone grafts. Patients underwent CT scans 1 week and 6 months after surgery for graft volume and density analysis. RESULTS: Two autologous and 3 fresh-frozen grafts failed. Autologous and fresh-frozen grafts lost, respectively, 28% and 46% of their initial volume (P = 0.028). It is noteworthy that less dense fresh-frozen blocks lost more volume than denser grafts (61% versus 16%). CONCLUSIONS: According to these 6-month results, only denser fresh-frozen bone graft may be an acceptable alternative to autologous bone for horizontal ridge augmentation. Further studies are needed to investigate its behaviour at longer time points.