Critical-sized Calvaria Defects Research Articles

Titanium-Enriched Hydroxyapatite–Gelatin Scaffolds with Osteogenically Differentiated Progenitor Cell Aggregates for Calvaria Bone Regeneration

Adequate bony support is the key to re-establish both function and esthetics in the craniofacial region. Autologous bone grafting has been the gold standard for regeneration of problematic large bone defects. However, poor graft availability and donor-site complications have led to alternative bone tissue-engineering approaches combining osteoinductive biomaterials and three-dimensional cell aggregates in scaffolds or constructs. The goal of the present study was to generate novel cell aggregate-loaded macroporous scaffolds combining the osteoinductive properties of titanium dioxide (TiO2) with hydroxyapatite-gelatin nanocomposites (HAP-GEL) for regeneration of craniofacial defects. Here we investigated the in vivo applicability of macroporous (TiO2)-enriched HAP-GEL scaffolds with undifferentiated and osteogenically differentiated multipotent adult progenitor cell (MAPC and OD-MAPC, respectively) aggregates for calvaria bone regeneration. The silane-coated HAP-GEL with and without TiO2 additives were polymerized and molded to produce macroporous scaffolds. Aggregates of the rat MAPC were precultured, loaded into each scaffold, and implanted to rat calvaria critical-size defects to study bone regeneration. Bone autografts were used as positive controls and a poly(lactic-co-glycolic acid) (PLGA) scaffold for comparison purposes. Preimplanted scaffolds and calvaria bone from pig were tested for ultimate compressive strength with an Instron 4411(®) and for porosity with microcomputerized tomography (μCT). Osteointegration and newly formed bone (NFB) were assessed by μCT and nondecalcified histology, and quantified by calcium fluorescence labeling. Results showed that the macroporous TiO2-HAP-GEL scaffold had a comparable strength relative to the natural calvaria bone (13.8±4.5 MPa and 24.5±8.3 MPa, respectively). Porosity was 1.52±0.8 mm and 0.64±0.4 mm for TiO2-HAP-GEL and calvaria bone, respectively. At 8 and 12 weeks postimplantation into rat calvaria defects, greater osteointegration and NFB were significantly present in the TiO2-enriched HAP-GEL constructs with OD-MAPCs, compared to the undifferentiated MAPC-loaded constructs, cell-free HAP-GEL with and without titanium, and PLGA scaffolds. The tissue-engineered TiO2-enriched HAP-GEL constructs with OD-MAPC aggregates present a potential useful therapeutic approach for calvaria bone regeneration.

Topical simvastatin improves the pro-angiogenic and pro-osteogenic properties of bioglass putty in the rat calvaria critical-size model

Abstract Objective: To describe the effect of bioactive glass putty with and without topical simvastatin on new bone formation in critical-sized defects of rat calvaria. Study design: A calvarial bone defect was created in 20 male Wistar rats and filled with bioactive glass alone (n=10) or combined with simvastatin (n=10). After 4 weeks, the defects were histomorphometrically evaluated for volume fraction (Vv) of woven bone, vessel density, bioglass quantity, and inflammation. Results: Compared to the bioglass-only group, rats treated with simvastatin had greater Vv of blood vessels (3.3%+0.7 vs 1.6%+0.1, P=0.0002) and new bone (2.3%+0.2 vs 1.8%+2.5, P=0.003). The Vv of the bioglass remnants in the bioglass-only group was higher than in the group treated with simvastatin (2.4% +0.08 vs 1.7%+0.3, P<0.0004). Chronic inflammation was noted in one rat from each group. Conclusion: Topical simvastatin seems to improve the pro-angiogenic and pro-osteogenic properties of bioglass putty in rat calvaria critical-size defects without significant inflammation.

Granule size-dependent bone regenerative capacity of octacalcium phosphate in collagen matrix.

The present study was designed to determine whether the osteoconductivity of octacalcium phosphate-collagen (OCP/Col) composite can be improved by controlling the granule size of OCP. The granules of synthetic OCP, with diameters in the range of 53 to 300, 300 to 500, and 500 to 1000 μm, were used as an inorganic source of composite materials mixed with atelo-Col. After vacuum dehydrothemal treatment, OCP/Col disks were implanted into critical-sized calvaria defects in Wistar rats for 4, 8, and 12 weeks and examined radiographically, histologically, histomorphometrically, and histochemically. The materials were characterized according to mercury intrusion porosimetry and scanning electron microscopy. X-ray diffraction was performed before and after implantation. The dissolution of OCP crystals in a Col matrix was determined by immersing OCP/Col disks in a culture medium. OCP/Col had a constant pore size (~30 μm) regardless of OCP granule size. OCP in the Col matrix tended to convert to hydroxyapatite (HA) during the implantation. OCP/Col with the smallest granules of OCP enhances both bone regeneration and biodegradation the most through tartrate-resistant acid phosphatase (TRAP)-positive osteoclastic cellular resorption of OCP granules. The smallest OCP granules in the Col matrix showed the highest dissolution and had the greatest potential to form HA. The results indicated that the size of the included OCP granules can controll the osteoconductivity of OCP/Col. The overall results suggest that the physicochemical property of OCP crystals is a factor that determines the bone regenerative capacity of OCP/Col in critical-sized calvaria large bone defects in rats.

The effect of synthetic octacalcium phosphate in a collagen scaffold on the osteogenicity of mesenchymal stem cells

Although the efficacy of the in vivo osteogenic capabilities of synthetic octacalcium phosphate (OCP) crystal implantation can be explained through its stimulatory capacity for the differentiation of the host osteoblastic cell lineage, direct evidence that OCP supports bone regeneration by osteogenic cells in vivo has not been shown. Mesenchymal stem cells (MSCs) isolated from 4-week-old male Wistar rat long bones were pre-incubated in osteogenic or maintenance medium in the presence or absence of basic fibroblast growth factor (bFGF). OCP/Collagen (OCP/Col) or collagen disks were seeded with MSCs that had been pre-incubated in osteogenic medium containing bFGF, which exhibited the highest differentiation induction, and then incubated for an additional day. The disks were implanted in critical-sized calvaria defects of 12-week-old male Wistar rats and the specimens were analysed radiographically, histologically, histomorphometrically, and by micro-computed tomography (CT) imaging at 4 and 8 weeks after the implantation. The OCP/Col·MSCs group rapidly induced more bone regeneration, even within 4 weeks, compared to the OCP/Col group without MSCs. The bone mineral density of the OCP/Col·MSCs group was also greater than the OCP/Col group. The Col·MSCs group did not exhibit prominent osteogenicity. These results indicate that OCP crystals in a collagen matrix efficiently promote exogenously introduced osteogenic cells to initiate bone regeneration if the cells are pre-treated in a suitable differentiation condition.

Craniofacial defect regeneration using engineered bone marrow mesenchymal stromal cells

Large craniofacial bony defects remain a significant clinical challenge. Bone marrow mesenchymal stromal cells (BM-MSCs) constitute a multipotent population. Previously, we developed a novel approach for BM-MSC expansion on 3D CultiSpher-S gelatin microcarrier beads in spin culture with preservation of their multipotentiality, reduction of apoptosis, and enhancement of bone formation in vivo. Here, we hypothesized that such cultured BM-MSCs without exogenous growth factors would respond to the orthopedic microenvironment, thus promoting craniofacial defect regeneration. BM-MSCs isolated from green fluorescent protein (GFP) transgenic rats were ex vivo expanded and transplanted into critical-sized (5-mm diameter) rat calvaria defects. Gelatin beads or defect alone served as controls. By 28 and 42 days, rats were sacrificed for microcomputed tomography (microCT), histologic, and immunohistochemistry examination. MicroCT results demonstrated that BM-MSCs were a statistically significant factor contributing to new bone volume regeneration. Histologic assessment showed that the BM-MSCs group produced more and higher quality new bone compared with beads or defect-alone groups in both osteoinductive and osteoconductive manners. Specifically, immunohistochemical staining identified GFP(+) cells residing in new bone lacunae in conjunction with non-GFP(+) cells. Therefore, ex vivo expanded BM-MSCs at least in part regenerated critical-sized calvaria defects by osteogenic differentiation in vivo.

Association of hyaluronic acid with a collagen scaffold may improve bone healing in critical-size bone defects

To evaluate the effects of a 1% hyaluronic acid (HA) gel in combination with an absorbable collagen sponge (ACS) in the healing of critical-size calvaria defects in rats. Thirty-two adult Wistar rats were used. Two 5-mm-diameter critical-size defects were created and the treatments were randomly distributed as follows: (1) 1% HA; (2) 1% HA gel-soaked ACS; (3) control (blood clot); and (4) ACS. The animals were sacrificed 60 days post-surgery, when biopsies were collected and processed for histology and histometric analysis. Bone fill was measured as the difference between the initial and the final defect sizes. Non-parametric tests were used to analyze differences between treatments (α=1%) and a t-test for body weight gain in each treatment group (α=5%). Histological analysis showed bone formation on the edges of the defects, although very limited, and a thin layer of connective tissue occupying the midportion of the defects in the control and the ACS groups. Defects filled with a 1% HA gel and 1% HA gel+ACS had a thicker layer of connective tissue and more new bone formed in the margins of the defects. Linear histometric measures showed no significant differences in the initial defect sizes between the groups (P>0.05). The association 1% HA gel+ACS (0.96 ± 0.14 mm) had significantly greater bone fill than the control (0.5 ± 0.02 mm) and ACS (0.56 ± 0.05 mm)-treated groups (P=0.0043 and 0.0173, respectively). Treatment with a 1% HA gel (0.7 ± 0.14 mm) showed no significant differences when compared with the other treatments. Within the limits of this study, a 1% HA gel associated with a collagen scaffold can improve new bone formation in critical-size defects. However, this treatment never resulted in complete closure of the defects and healing in the major portion of the defects was characterized by fibrous tissue.

The effect of BMP-2 on the osteoconductive properties of β-tricalcium phosphate in rat calvaria defects

Bone formation in critical-sized calvaria defects is strongly dependent on the osteoconductive properties of grafts. It remains a matter of controversy whether biomaterials can replace autografts and whether the supplementation of biomaterials with Bone Morphogenetic Proteins (BMPs) is necessary to enhance bone formation. We examined rat calvaria critical-sized defects (5-mm-diameter) treated with β-tricalcium phosphate (TCP; Cerasorb ® M), polylactic and polyglycolic acid gel (PLA/PGA; Fisiograft ®) and calcium phosphate cement (CPC; Norian ® CRS ®), either alone or in the presence of 5 μg of BMP-2 after 45 days. Autografts and untreated defects served as controls. Bone formation was evaluated based on μCT analysis, histomorphometric analysis and fluorescence analysis. We report that TCP supported bone formation more efficiently than did autografts. Bone formation in the presence of TCP alone reached a maximal level, as BMP-2 supplementation failed to enhance bone formation. By contrast, no significant difference in bone formation was observed when PLA/PGA and CPC were compared to autografts. Moreover, the presence of BMP-2 did not substantially change the osteoconductive properties of PLA/PGA or CPC. We conclude that the osteoconductive properties of TCP are superior to those of autografts and that TCP does not require BMP-2 supplementation. Our findings also show that the decreased osteoconductive properties of PLA/PGA and CPC cannot be overcome by BMP-2 supplementation in rat calvaria defects.

Octacalcium Phosphate–Precipitated Alginate Scaffold for Bone Regeneration

The present study was designed to investigate whether octacalcium phosphate (OCP)-precipitated alginate (Alg) promotes osteoblastic cell proliferation and bone regeneration in comparison with Alg itself. Alg, known to lack mammal cell attachment capability, was used as the matrix to test the distribution effect of OCP in a three-dimensional environment. A series of Alg/OCP scaffolds with different pore sizes was prepared by centrifuging Alg gels precipitated by OCP crystals. The scaffolds had a bimodal distribution of pores (ultrafine pores: approximately 100 nm; relatively large pores: from 6.0 to 51.7 microm) and over 86% porosity. The osteoconductive capability of Alg/OCP was determined by examining mouse bone marrow stromal ST-2 cell proliferation after 3 days in vitro and bone regeneration in mouse calvaria critical-sized defect after 21 days. The analyses showed that ST-2 cell proliferation and bone regeneration increased with an increase in the pore size and reached the highest level in the 51.7 microm pore scaffold. The results suggest that OCP-precipitated Alg provides a better scaffold for osteoblasts to attach and proliferate in a three-dimensional environment and promotes bone regeneration, indicating that OCP is a candidate material to modify the surface of non-cell-interactive polymeric scaffolds, such as Alg, into an osteogenic condition.

Comparative study on bone regeneration by synthetic octacalcium phosphate with various granule sizes

The present study was designed to investigate whether the granule size of synthetic octacalcium phosphate (OCP) and the resultant intergranular spaces between the granules formed by the filling affect its osteoconductive and biodegradable characteristics in a mouse calvaria critical-sized defect up to 10 weeks after implantation. Mercury intrusion porosimetry showed that OCP granules having distinct diameter sizes ranging from 53 to 300 (S-OCP), 300 to 500 (I-OCP) and 500 to 1000 μm (L-OCP) produced distinct intergranular spaces between OCP granules ranging from 28.8 to 176.6 μm. The dissolution rate of OCP, estimated by the phosphate concentration in the culture medium, was the highest in S-OCP, followed by I-OCP and L-OCP, while the specific surface area of OCP decreased. Histological and histomorphometric analyses showed that bone formation around the implanted granules increased significantly with increasing granule size coupled with activating the appearance of TRAP- and cathepsin K-positive osteoclastic cells. The rate of new bone formation formed with L-OCP was two times higher than that formed with S-OCP at 10 weeks after implantation. The results indicated that the osteoconductive and biodegradable properties of OCP can be augmented by increasing the granule size, most probably by thus providing enough spaces between the granules, suggesting that the intergranular spaces formed by the granules may work similarly to pores, as reported in porous ceramic materials. It seems likely that the enhancement of bone formation by OCP is accompanied by simultaneous activation of osteoclastic resorption of OCP.

Natural Bovine Anorganic Apatite and Collagen Presents Osteoconductivity and Contribute to Bone Repair of Rat Calvaria Critical Size Defect

The aim of this study was verify the biological efficacy of the use of a xenograft for bone loss therapy. Blood clot, particulate autogenous bone or anorganic bovine xenograft filled critical size defects (CSD) in rat calvaria (8mm diameter). After 0, 7, 30 and 90 days the animals were killed and macroscopic, radiographic and histopathological analysis were conducted. Although no treatment promoted the total closure of bone defect, autogenous bone group had better bone repair after 90 days, followed by xenograft group that exhibited direct bone neoformation onto, and around, the particles confirming its osteoconductivity. In conclusion, the xenograft tested in vivo showed biocompatibility, biodegradability and osteoconductive properties in rat calvaria CSD.

Calcium Sulfate and Platelet-Rich Plasma make a novel osteoinductive biomaterial for bone regeneration

BackgroundWith the present study we introduce a novel and simple biomaterial able to induce regeneration of bone. We theorized that nourishing a bone defect with calcium and with a large amount of activated platelets may initiate a series of biological processes that culminate in bone regeneration. Thus, we engineered CS-Platelet, a biomaterial based on the combination of Calcium Sulfate and Platelet-Rich Plasma in which Calcium Sulfate also acts as an activator of the platelets, therefore avoiding the need to activate the platelets with an agonist.MethodsFirst, we tested CS-Platelet in heterotopic (muscle) and orthotopic (bone) bone regeneration bioassays. We then utilized CS-Platelet in a variety of dental and craniofacial clinical cases, where regeneration of bone was needed.ResultsThe heterotopic bioassay showed formation of bone within the muscular tissue at the site of the implantation of CS-Platelet. Results of a quantitative orthotopic bioassay based on the rat calvaria critical size defect showed that only CS-Platelet and recombinant human BMP2 were able to induce a significant regeneration of bone. A non-human primate orthotopic bioassay also showed that CS-Platelet is completely resorbable. In all human clinical cases where CS-Platelet was used, a complete bone repair was achieved.ConclusionThis study showed that CS-Platelet is a novel biomaterial able to induce formation of bone in heterotopic and orthotopic sites, in orthotopic critical size bone defects, and in various clinical situations. The discovery of CS-Platelet may represent a cost-effective breakthrough in bone regenerative therapy and an alternative or an adjuvant to the current treatments.

Effects of Runx2 genetic engineering and in vitro maturation of tissue‐engineered constructs on the repair of critical size bone defects

Genetic and tissue engineering strategies are being pursued to address the clinical limitations of current bone grafting materials. Based on our previous work demonstrating that overexpression of the Runx2 osteoblastic transcription factor and in vitro construct maturation synergistically enhanced in vivo mineralization in an ectopic site (Byers et al., Tissue Eng 2004;10:1757-1766), we examined the effects of these two parameters on the repair of critical size bone defects. Primary rat bone marrow stromal cells transduced with Runx2 or control (no Runx2 insert) retroviral vector were seeded onto 3D fused deposition-modeled polycaprolactone scaffolds. Runx2-modified cells produced biologically-equivalent mineralized matrices at nearly 2-fold higher rates than control cells. Constructs cultured in vitro for 1 day (immature) or 21 days (mineralized) were subsequently implanted into critical size calvaria defects in syngeneic rats, and bone healing was analyzed by micro-CT and histomorphometry at 28 days. Runx2-modified and control constructs precultured for 1 day healed to a greater extent than defects receiving no implant. Cell-free scaffolds yielded equivalent levels of bone formation as constructs precultured for 1 day. Interestingly, defects treated with control cell-seeded constructs precultured for 21 days exhibited low bone formation compared to other construct treatments, and repair was comparable to empty defects. In contrast, Runx2-modified constructs precultured for 21 days contained twice as much bone as control constructs precultured for 21 days and equivalent levels of new bone as cell-free and 1 day precultured constructs. These results demonstrate interplay between Runx2 genetically-modified cells and in vitro construct maturation in bone healing responses.

Effect of platelet-rich-plasma on cranial distraction osteogenesis in sheep: preliminary clinical and radiographic results

The purpose of this study was to investigate the effect of platelet-rich-plasma (PRP) on cranial distraction osteogenesis. Standardized calvaria critical size defects (6 cm × 5 cm) were created in 16 adult female sheep. Bifocal cranial transport distraction osteogenesis with autogenous free calvaria bone grafts (2 cm × 4 cm) was performed at a rate of 1 mm once daily to a total of 30 mm. The 16 sheep involved in the experiment were randomly divided into four groups, four animals in each: Group 1 (no PRP, latency 5 days); Group 2 (no PRP, latency 0 day), Group 3 (PRP, latency 5 days) and Group 4 (PRP, latency 0 day). After a consolidation phase of 6 weeks, the animals were sacrificed and specimens harvested for conventional radiological and 3D quantitative computer tomographic (3D-QCT) assessment. New bone was generated in the distraction zone in all groups. There were significantly ( P < 0.05) higher densities in the proximal region of the distraction regenerate in Group 4 (PRP, latency 0 day) compared to Group 2. However, no significant differences in mean density of the total distraction regenerate were found, neither in volume of the bony regenerate between the experimental groups. This study showed that PRP only had an effect on bone regeneration if active distraction was started immediately after application of PRP in the distraction gap.

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part II. Bioengineering implants to optimize bone replacement in reconstruction of cranial defects.

The present study investigated hydroxyapatite biomaterials implanted in critical-size defects in the calvaria of adult sheep to determine the optimal bioengineering of hydroxyapatite composites to facilitate bone ingrowth into these materials. Five calvarial defects measuring 16.8 mm in diameter were made in each of 10 adult sheep. Three defects were filled with cement paste composites of hydroxyapatite and beta-tricalcium phosphate as follows: (1) 100 percent hydroxyapatite-cement paste, (2) 60 percent hydroxyapatite-cement paste, and (3) 20 percent hydroxyapatite-cement paste. One defect was filled with a ceramic composite containing 60 percent hydroxyapatite-ceramic, and the fifth defect remained unfilled. One year after implantation, the volume of all biomaterials was determined by computed tomography, and porosity and bone replacement were determined using backscatter electron microscopy. Computed tomography-based volumetric assessment 1 year after implantation demonstrated that none of the unfilled cranial defects closed over the 1-year period, confirming that these were critical-size defects. There was a significant increase in volume in both the cement paste and ceramic implants containing 60 percent hydroxyapatite (p < 0.01). There was no significant change in volume of the remaining cement paste biomaterials. Analysis of specimens by backscatter electron microscopy demonstrated mean bone replacement of 4.8 +/- 1.4 percent (mean +/- SEM) in 100 percent hydroxyapatite-cement paste, 11.2 +/- 2.3 percent in 60 percent hydroxyapatite-cement paste, and 28.5 +/- 4.5 percent in 20 percent hydroxyapatite-cement paste. There was an inverse correlation between the concentration of hydroxyapatite and the amount of bone replacement in the cement paste for each composite tested (p < 0.01). Bone replacement in 60 percent hydroxyapatite-ceramic composite (13.6 +/- 2.0 percent) was not significantly different from that in 60 percent hydroxyapatite-cement paste. Of note is that the ceramic composite contained macropores (200 to 300 microm) that did not change in size over the 1-year period. All cement paste composites initially contained micropores (3 to 5 nm), which remained unchanged in 100 percent hydroxyapatite-cement paste. Cement paste implants containing increased tricalcium phosphate demonstrated a corresponding increase in macropores following resorption of the tricalcium phosphate component. Bone replacement occurred within the macropores of these implants. In conclusion, there was no significant bone ingrowth into pure hydroxyapatite-cement paste (Bone Source, Stryker-Leibinger Inc., Dallas, Texas) in the present study. The introduction of macropores in a biomaterial can optimize bone ingrowth for reconstruction of critical-size defects in calvaria. This was demonstrated in both the ceramic composite of hydroxyapatite tested and the cement paste composites of hydroxyapatite by increasing the composition of a rapidly resorbing component such as beta-tricalcium phosphate.

The Osteogenic Potential of Two Composite Graft Systems Using Osteogenin

The purpose of this study was to determine the quantity of new bone formation in critical sized calvaria defects in rats treated with two composite graft systems. The systems consisted of either a combination of the bone inductive protein (osteogenin) plus type I collagen (Os + C) or the combination of osteogenin with coralline hydroxyapatite (Os + HA). Additional treatments consisted of coralline hydroxyapatite (HA) or untreated control defects. After 28 days the calvaria were recovered and processed for quantitative radiography (radiomorphometry) and histomorphometry. Histomorphometric results were based on quantitation of regenerated trabecular bone. Results indicated that the Os + C combination produced substantially more bone than the Os + HA, HA, or control groups (P less than 0.05). Radiomorphometric assessment was based on the detection of radiopacity in the calvarial wounds. Due to the radiopaque property of HA, it was not possible to accurately quantitate the radiopacity of the regenerating bone from HA and host bone. Therefore, conclusions about the efficacy of the treatments must be derived from histomorphometric data. Results from histometric measurements of healing indicate that the Os + C combination has the greatest potential for regenerating calvarial bone defects. The potential for osteogenin in regenerating alveolar bone lost due to periodontal disease is suggested by these studies.