Biphasic Calcium Phosphate Granules Research Articles

Fabrication of magnesium-doped biphasic calcium phosphate granules with sea urchin spine-derived porous structure

In bone regeneration, synthetic materials are required to replace autogenous bone grafts. The shape, composition, surface, and porous structure of the material are key factors in bone formation and material resorption. In this study, we describe the fabrication of Mg-doped biphasic calcium phosphate (Mg-BCP) granules comprising hydroxyapatite (HAp) and beta-tricalcium phosphate (β-TCP) by exploiting the structural and chemical characteristics of sea urchin spines. The Mg-BCP granules were cylinders with a 1.2–1.8 mm diameter and height of 1.4–1.7 mm. The β-TCP/HAp ratio and Mg content in the Mg-BCP granules were controlled within the ranges of 0.36–0.45 and 2.40–6.21 mol%, respectively. Controlling the β-TCP/HAp ratio and Mg content may contribute to controlling the material resorption rate and promoting osteogenesis and angiogenesis. The Mg-BCP granules maintained the structural characteristics of the sea urchin spines. The granules comprised radial wedges termed septa, and a meshwork termed stereom. The interseptal gaps, or micropores, decreased from the granular exterior (24–29 μm) to the interior (14–28 μm). The septa in the outermost layer provide concavities on the sides of the cylindrical granules. The stereom meshworks in the core and outer layers involved micropores in the ranges of 3–15 and 3–10 μm, respectively. These multiscale pores and surface undulations may improve cell penetration and attachment, bodily fluid permeability, and protein adsorption. The compressive strengths of the Mg-BCP granules were in the range of 45.2–46.1 MPa, which are useful as bone grafts. Due to their rounded shapes, the Mg-BCP granules filled a mold more densely than the clinically used and irregularly shaped granules. These findings demonstrated that Mg-BCP granules are promising materials for bone regeneration.

EVALUATION OF A CALCIUM PHOSPHATE-COLLAGEN MATRIX BONE GRAFT WITH NEEDLE-SHAPED SUBMICRON SURFACE TOPOGRAPHY IN A CLINICALLY RELEVANT SHEEP POSTEROLATERAL LUMBAR SPINE FUSION MODEL

Biphasic calcium phosphate (BCP) with a characteristic needle-shaped submicron surface topography (MagnetOs) has attracted much attention due to its unique bone-forming ability which is essential for repairing critical-size bone defects such as those found in the posterolateral spine. Previous in vitro and ex-vivo data performed by van Dijk LA and Yuan H demonstrated that these specific surface characteristics drive a favorable response from the innate immune system.This study aimed to evaluate and compare the in vivo performance of three commercially-available synthetic bone grafts, (1) i-FACTOR Putty®, (2) OssDsign® Catalyst Putty and (3) FIBERGRAFT® BG Matrix, with that of a novel synthetic bone graft in a clinically-relevant instrumented sheep posterolateral lumbar spine fusion (PLF) model. The novel synthetic bone graft comprised of BCP granules with a needle-shaped submicron surface topography (MagnetOs) embedded in a highly porous and fibrillar collagen matrix (MagnetOs Flex Matrix).Four synthetic bone grafts were implanted as standalone in an instrumented sheep PLF model for 12 weeks (n=3 bilateral levels per group; levels L2/3 & L4/5), after which spinal fusion was determined by manual palpation, radiograph and µCT imaging (based on the Lenke scale), range-of-motion mechanical testing, and histological and histomorphological evaluation.Radiographic fusion assessment determined bilateral robust bone bridging (Lenke scale A) in 3/3 levels for MagnetOs Flex Matrix compared to 1/3 for all other groups. For µCT, bilateral fusion (Lenke scale A) was found in 2/3 levels for MagnetOs Flex Matrix, compared to 0/3 for i-FACTOR Putty®, 1/3 for OssDsign® Catalyst Putty and 0/3 for FIBERGRAFT® BG Matrix. Fusion assessment for MagnetOs Flex Matrix was further substantiated by histology which revealed significant graft resorption complemented by abundant bone tissue and continuous bony bridging between vertebral transverse processes resulting in bilateral spinal fusion in 3/3 implants.These results show that MagnetOs Flex Matrix achieved better fusion rates compared to three commercially-available synthetic bone grafts when used as a standalone in a clinically-relevant instrumented sheep PLF model.

Injectable Hydrogel Membrane for Guided Bone Regeneration.

In recent years, multicomponent hydrogels such as interpenetrating polymer networks (IPNs) have emerged as innovative biomaterials due to the synergistic combination of the properties of each network. We hypothesized that an innovative non-animal IPN hydrogel combining self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) with photochemically cross-linkable dextran methacrylate (DexMA) could be a valid alternative to porcine collagen membranes in guided bone regeneration. Calvaria critical-size defects in rabbits were filled with synthetic biphasic calcium phosphate granules in conjunction with Si-HPMC; DexMA; or Si-HPMC/DexMA experimental membranes; and in a control group with a porcine collagen membrane. The synergistic effect obtained by interpenetration of the two polymer networks improved the physicochemical properties, and the gel point under visible light was reached instantaneously. Neutral red staining of murine L929 fibroblasts confirmed the cytocompatibility of the IPN. At 8 weeks, the photo-crosslinked membranes induced a similar degree of mineral deposition in the calvaria defects compared to the positive control, with 30.5 ± 5.2% for the IPN and 34.3 ± 8.2% for the collagen membrane. The barrier effect appeared to be similar in the IPN test group compared with the collagen membrane. In conclusion, this novel, easy-to-handle and apply, photochemically cross-linkable IPN hydrogel is an excellent non-animal alternative to porcine collagen membrane in guided bone regeneration procedures.

Preparation and evaluation of osteoinductive porous biphasic calcium phosphate granules obtained from eggshell for bone tissue engineering

The synthetic bone graft substitutes currently used clinically are osteoconductive but not osteoinductive; their low success rate is thus their biggest disadvantage. The use of biomass raw materials for synthesizing calcium phosphate has gradually attracted increased research attention. In this study, hydroxyapatite powder was prepared through liquid-phase precipitation from eggshell, and porous biphasic calcium phosphate granules (EBGs) were then obtained using a pore former and sintering procedure. The EBGs were discovered to have high biocompatibility and no cytotoxicity. The results of animal experiments showed that the area of new bone growth was high, numerous Haversiancanals could be observed, and almost all EBGs were surrounded by new bone tissue, which proved that the EBGs had excellent osteoinductivity. By contrast, numerous fat cells were found in the femoral defect area when a commercial bone graft was employed. Various biological inorganic ions (Mg, Sr, Na, and Fe) originally in the eggshell raw materials were incorporated into the EBGs, and the EBGs exhibited excellent osteogenic abilities. The developed approach provides an economical and feasible solution for the treatment of bone defects.

A Comparative In Vitro and In Vivo Study of Osteogenicity by Using Two Biomaterials and Two Human Mesenchymal Stem Cell Subtypes.

Background: Bone repair induced by stem cells and biomaterials may represent an alternative to autologous bone grafting. Mesenchymal stromal/stem cells (MSCs), easily accessible in every human, are prototypical cells that can be tested, alone or with a biomaterial, for creating new osteoblasts. The aim of this study was to compare the efficiency of two biomaterials—biphasic calcium phosphate (BCP) and bioactive glass (BG)—when loaded with either adult bone marrow mesenchymal stem cells (BMMSCs) or newborn nasal ecto-mesenchymal stem cells (NE-MSCs), the latter being collected for further repair of lip cleft-associated bone loss. Materials and Methods: BMMSCs were collected from two adults and NE-MSCs from two newborn infants. An in vitro study was performed in order to determine the best experimental conditions for adhesion, viability, proliferation and osteoblastic differentiation on BCP or BG granules. Bone-associated morphological changes and gene expression modifications were quantified using histological and molecular techniques. The in vivo study was based on the subcutaneous implantation in nude mice of the biomaterials, loaded or not with one of the two cell types. Eight weeks after, bone formation was assessed using histological and electron microscopy techniques. Results: Both cell types—BMMSC and NE-MSC—display the typical stem cell surface markers—CD73+, CD90+, CD105+, nestin - and exhibit the MSC-associated osteogenic, chondrogenic and adipogenic multipotency. NE-MSCs produce less collagen and alkaline phosphatase than BMMSCs. At the transcript level, NE-MSCs express more abundantly three genes coding for bone sialoprotein, osteocalcin and osteopontin while BMMSCs produce extra copies of RunX2. BMMSCs and NE-MSCs adhere and survive on BCP and BG. In vivo experiments reveal that bone formation is only observed with BMMSCs transplanted on BCP biomaterial. Conclusion: Although belonging to the same superfamily of mesenchymal stem cells, BMMSCs and NE-MSCs exhibit striking differences, in vitro and in vivo. For future clinical applications, the association of BMMSCs with BCP biomaterial seems to be the most promising.

Bilateral double site (calvarial and mandibular) critical-size bone defect model in rabbits for evaluation of a craniofacial tissue engineering constructs.

Most existing preclinical models for evaluating the biosafety and bone-regeneration efficacy of innovative bone substitute materials (BSMs) or tissue engineering (TE) constructs only consisted of a single-site defect and the anatomical locations of defect varied drastically. While the compelling evidence showed that the bone healing pattern is location-dependent, owing to developmental, structural, and functional differences of anatomical locations, this is particularly true for the craniofacial region. Taking this into account, the bone healing efficiency of a BSM shown at one anatomical defect location cannot ensure the same impact at another. This prompted us to develop, for the first time, a model of bilateral critical-sized defect (CSD) at two distinctly different locations (non-load-bearing parietal calvaria and load-bearing mandibular body) co-existing in one rabbit to reduce the number of animals needed and avoid the influence of interindividual variability and evaluation bias on comparisons.24 healthy adult male New Zealand White rabbits were randomly assigned to a group, either control, autograft (considered the “gold standard”) or a clinically relevant BSM (biphasic calcium phosphate granules) (BCPg, Mastergraft®, Medronics). The full-thickness cylindrical calvarial defect (ø10 mm) on frontoparietal region and mandibular composite defect (ø11 mm) on the body of the mandible were created bilaterally using low-speed drilling with saline irrigation. The defect on one side was filled with autograft debris or BCPg, and the other side was no graft (empty). Following the euthanasia of animals at the predetermined intervals (4w and 12w), the defect zones were examined macroscopically and then sampled and processed for microcomputed tomography (microCT) and histological analysis.All surgeries went uneventfully, and all rabbits recovered slowly but steadily. No symptoms of infection or inflammation associated with the defect were observed during the experiment. At 4w and 12w, macroscopic views of all defect sites were clean without any signs of necrosis or abscess, and no intraoral communication was found. The analysis of microCT and histological findings showed the non-healing nature of the empty defect, thereby both calvaria and mandible CSDs can be validated. The study of the application of BCPg in this defect model highlighted good osteointegration and excellent osteoconductive properties but compromised the osteoinductive properties of this material (compared with autograft).To conclude, this novel double-site CSD model holds great promise in the application for preclinical evaluation of BSMs, TE construct, etc. With a reduced number of animals in use, and lower interindividual variability and evaluation bias for comparisons.

Biologically Relevant In Vitro 3D-Model to Study Bone Regeneration Potential of Human Adipose Stem Cells.

Standard cell cultures may not predict the proliferation and differentiation potential of human mesenchymal stromal cells (MSCs) after seeding on a scaffold and implanting this construct in a bone defect. We aimed to develop a more biologically relevant in vitro 3D-model for preclinical studies on the bone regeneration potential of MSCs. Human adipose tissue-derived mesenchymal stromal cells (hASCs; five donors) were seeded on biphasic calcium phosphate (BCP) granules and cultured under hypoxia (1% O2) for 14 days with pro-inflammatory TNFα, IL4, IL6, and IL17F (10 mg/mL each) added during the first three days, simulating the early stages of repair (bone construct model). Alternatively, hASCs were cultured on plastic, under 20% O2 and without cytokines for 14 days (standard cell culture). After two days, the bone construct model decreased total DNA (3.9-fold), COL1 (9.8-fold), and RUNX2 expression (19.6-fold) and metabolic activity (4.6-fold), but increased VEGF165 expression (38.6-fold) in hASCs compared to standard cultures. After seven days, the bone construct model decreased RUNX2 expression (64-fold) and metabolic activity (2.3-fold), but increased VEGF165 (54.5-fold) and KI67 expression (5.7-fold) in hASCs compared to standard cultures. The effect of the bone construct model on hASC proliferation and metabolic activity could be largely mimicked by culturing on BCP alone (20% O2, no cytokines). The effect of the bone construct model on VEGF165 expression could be mimicked by culturing hASCs under hypoxia alone (plastic, no cytokines). In conclusion, we developed a new, biologically relevant in vitro 3D-model to study the bone regeneration potential of MSCs. Our model is likely more suitable for the screening of novel factors to enhance bone regeneration than standard cell cultures.

Porous Biphasic Calcium Phosphate Granules from Oyster Shell Promote the Differentiation of Induced Pluripotent Stem Cells.

Oyster shells are rich in calcium, and thus, the potential use of waste shells is in the production of calcium phosphate (CaP) minerals for osteopathic biomedical applications, such as scaffolds for bone regeneration. Implanted scaffolds should stimulate the differentiation of induced pluripotent stem cells (iPSCs) into osteoblasts. In this study, oyster shells were used to produce nano-grade hydroxyapatite (HA) powder by the liquid-phase precipitation. Then, biphasic CaP (BCP) bioceramics with two different phase ratios were obtained by the foaming of HA nanopowders and sintering by two different two-stage heat treatment processes. The different sintering conditions yielded differences in structure and morphology of the BCPs, as determined by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Brunauer–Emmett–Teller (BET) surface area analysis. We then set out to determine which of these materials were most biocompatible, by co-culturing with iPSCs and examining the gene expression in molecular pathways involved in self-renewal and differentiation of iPSCs. We found that sintering for a shorter time at higher temperatures gave higher expression levels of markers for proliferation and (early) differentiation of the osteoblast. The differences in biocompatibility may be related to a more hierarchical pore structure (micropores within macropores) obtained with briefer, high-temperature sintering.

The effectiveness of hydroxyapatite-beta tricalcium phosphate incorporated into stem cells from human exfoliated deciduous teeth for reconstruction of rat calvarial bone defects.

To investigate the effects of stem cells from the pulp of human exfoliated deciduous teeth (SHED) on biphasic calcium phosphate granules (BCP) to repair rat calvarial defects as compared to autogenous bone grafting. A defect with a 6-mm diameter was produced on the calvaria of 50 rats. BCP granules were incorporated into SHED cultures grown for 7 days in conventional (CM) or osteogenic (OM) culture media. The animals were allocated into 5 groups of 10, namely: clot, autogenous bone, BCP, BCP+SHED in CM (BCP-CM), and BCP+SHED in OM (BCP-OM). The presence of newly formed bone and residual biomaterial particles was assessed by histometric analysis after 4 and 8 weeks. The autogenous group showed the largest newly formed bone area at week 8 and in the entire experimental period, with a significant difference in relation to the other groups (P<0.05). At week 8, BCP-CM and BCP-OM groups showed homogeneous new bone formation (P=0.13). When considering the entire experimental period, the BCP group had the highest percentage of residual particle area, with no significant difference from the BCP-CM group (P=0.06) and with a significant difference from the BCP-OM group (P=0.01). BCP-CM and BCP-OM groups were homogeneous throughout the experimental period (P=0.59). BCP incorporated into SHED cultures showed promising outcomes, albeit less pronounced than autogenous grafting, for the repair of rat calvarial defects. BCP incorporated into SHED cultures showed to be an alternative in view of the disadvantages to obtain autogenous bone graft.

Plant-derived soybean peroxidase stimulates osteoblast collagen biosynthesis, matrix mineralization, and accelerates bone regeneration in a sheep model

Bone defects arising from fractures or disease represent a significant problem for surgeons to manage and are a substantial economic burden on the healthcare economy. Recent advances in the development of biomaterial substitutes provides an attractive alternative to the current “gold standard” autologous bone grafting. Despite on-going research, we are yet to identify cost effective biocompatible, osteo-inductive factors that stimulate controlled, accelerated bone regeneration.We have recently reported that enzymes with peroxidase activity possess previously unrecognised roles in extracellular matrix biosynthesis, angiogenesis and osteoclastogenesis, which are essential processes in bone remodelling and repair. Here, we report for the first time, that plant-derived soybean peroxidase (SBP) possesses pro-osteogenic ability by promoting collagen I biosynthesis and matrix mineralization of human osteoblasts in vitro. Mechanistically, SBP regulates osteogenic genes responsible for inflammation, extracellular matrix remodelling and ossification, which are necessary for normal bone healing. Furthermore, SBP was shown to have osteo-inductive properties, that when combined with commercially available biphasic calcium phosphate (BCP) granules can accelerate bone repair in a critical size long bone defect ovine model. Micro-CT analysis showed that SBP when combined with commercially available biphasic calcium phosphate (BCP) granules significantly increased bone formation within the defects as early as 4 weeks compared to BCP alone. Histomorphometric assessment demonstrated accelerated bone formation prominent at the defect margins and surrounding individual BCP granules, with evidence of intramembranous ossification. These results highlight the capacity of SBP to be an effective regulator of osteoblastic function and may be beneficial as a new and cost effective osteo-inductive agent to accelerate repair of large bone defects.

Ex Vivo and In Vivo Analyses of Novel 3D-Printed Bone Substitute Scaffolds Incorporating Biphasic Calcium Phosphate Granules for Bone Regeneration.

(1) Background: The aim of this study was examining the ex vivo and in vivo properties of a composite made from polycaprolactone (PCL) and biphasic calcium phosphate (BCP) (synprint, ScientiFY GmbH) fabricated via fused deposition modelling (FDM); (2) Methods: Scaffolds were tested ex vivo for their mechanical properties using porous and solid designs. Subcutaneous implantation model analyzed the biocompatibility of PCL + BCP and PCL scaffolds. Calvaria implantation model analyzed the osteoconductive properties of PCL and PCL + BCP scaffolds compared to BCP as control group. Established histological, histopathological and histomorphometrical methods were performed to evaluate new bone formation.; (3) Results Mechanical testing demonstrated no significant differences between PCL and PCL + BCP for both designs. Similar biocompatibility was observed subcutaneously for PCL and PCL + BCP scaffolds. In the calvaria model, new bone formation was observed for all groups with largest new bone formation in the BCP group, followed by the PCL + BCP group, and the PCL group. This finding was influenced by the initial volume of biomaterial implanted and remaining volume after 90 days. All materials showed osteoconductive properties and PCL + BCP tailored the tissue responses towards higher cellular biodegradability. Moreover, this material combination led to a reduced swelling in PCL + BCP; (4) Conclusions: Altogether, the results show that the newly developed composite is biocompatible and leads to successful osteoconductive bone regeneration. The new biomaterial combines the structural stability provided by PCL with bioactive characteristics of BCP-based BSM. 3D-printed BSM provides an integration behavior in accordance with the concept of guided bone regeneration (GBR) by directing new bone growth for proper function and restoration.

Injectable silanized hyaluronic acid hydrogel/biphasic calcium phosphate granule composites with improved handling and biodegradability promote bone regeneration in rabbits.

Biphasic calcium phosphate (BCP) granules are osteoconductive biomaterials used in clinics to favor bone reconstruction. Yet, poor cohesivity, injectability and mechanical properties restrain their use as bone fillers. In this study, we incorporated BCP granules into in situ forming silanized hyaluronic acid (Si-HA) and hydroxypropylmethylcellulose (Si-HPMC) hydrogels. Hydrogel composites were shown to be easily injectable (F < 30 N), with fast hardening properties (<5 min), and similar mechanical properties (E∼ 60 kPa). In vivo, both hydrogels were well tolerated by the host, but showed different biodegradability with Si-HA gels being partially degraded after 21d, while Si-HPMC gels remained stable. Both composites were easily injected into critical size rabbit defects and remained cohesive. After 4 weeks, Si-HPMC/BCP led to poor bone healing due to a lack of degradation. Conversely, Si-HA/BCP composites were fully degraded and beneficially influenced bone regeneration by increasing the space available for bone ingrowth, and by accelerating BCP granules turnover. Our study demonstrates that the degradation rate is key to control bone regeneration and that Si-HA/BCP composites are promising biomaterials to regenerate bone defects.

In Vitro and In Vivo Evaluation of Nanostructured Biphasic Calcium Phosphate in Granules and Putty Configurations.

Synthetic biphasic calcium phosphate (BCP) granules and powder are biocompatible biomaterials with a well-known capacity for osteoconduction, presenting very satisfactory clinical and histological results. It remains unanswered if the putty configuration impacts the biological response to the material. In this study, we aimed to compare the cytocompatibility and biocompatibility of nanostructured BCP in the putty configuration (moldable nanostructured calcium phosphate, MnCaP) on the healing of critical-sized bone defects (8 mm) in rat calvaria. Cytocompatibility was determined through the viability of fibroblast cells (V-79) to the extracts of different concentrations of MnCaP. Forty-five Wistar rats were randomly divided into three groups (n = 15)—clot, MnCaP, and commercial biphasic calcium phosphate in granules configurations (Nanosynt®)—and subdivided into three experimental periods (1, 3, and 6 months). Histological, histomorphometric, and microtomographic analyses allowed the evaluation of newly formed bone, residual biomaterial, and connective tissue. The in vitro evaluation showed that MnCaP was cytocompatible. The histomorphometric results showed that the Nanosynt® group granted the highest new-formed bone values at six months (p < 0.05), although the biomaterial volume did not differ between groups. The putty configuration was easier to handle, and both configurations were biocompatible and osteoconductive, presented similar biosorption rates, and preserved the calvaria architecture.

Evaluation of bone healing of odontogenic maxillary cystic defects grafted with hemihydrated calcium sulphate granules Versus biphasic calcium phosphate granules: A Randomized Controlled Clinical Trial

Objectives: This study aims to compare between the effect of hemihydrated calcium sulphate bone graft granules and biphasic calcium phosphate granules on bone healing of maxillary cystic defects. Patients and methods: Twenty patients (15 males, 5 females; age range, 16 to 40 years with an average of 25 years) with maxillary cystic lesions were included in this prospective, comparative study. Patients were randomized into two groups as group 1 in which the cystic defects were grafted with hemihydrated calcium sulphate bone graft granules (n=10) and group 2 in which the cystic defects were treated with biphasic calcium phosphate granules(SYMBIOS) (n=10). Postoperative assessments were performed for both groups, clinically through detection of any signs of infection, inflammation, or graft rejection; and radiographically. Radiographic assessment was performed by using cone beam computed tomography (CBCT) preoperative and at 6 months postoperatively for measurement of relative bone density and dimensional changes of the cystic cavity. Results: The radiographic results demonstrated a statistical insignificant difference between the medians (IQR) of relative dimension and density changes of cystic defects in both groups after 6 months. Conclusion: Both bone grafts granules have the same effects on bone filling of cystic defects. Also, both grafts are similar in reducing dimensions of cystic cavities during healing.

Development of injectable chitosan/biphasic calcium phosphate bone cement and in vitro and in vivo evaluation

Injectable biphasic calcium phosphate bone cements (BCPCs) composed of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) have been intensively investigated because of their high rate of biodegradation, bioactivity and osteoconductivity, which can be adjusted by changing the ratio between β-TCP and HA phases after setting. The aim of this study was to evaluate the performance of 1 wt% chitosan fiber additive with biphasic calcium phosphate as an injectable bone cement both in vitro and in vivo. In vitro evaluation of compressive strength, degradation rate, morphology, and cell and alkaline phosphatase activities was done by comparison with bone cement without β-TCP. The in vivo results for micro-CT scanning and histological examinations for three groups (control, BCPC and commercial biphasic calcium phosphate granules) were characterized and compared. After the addition of 20 wt% β-TCP to calcium phosphate cement, the initial and final setting times of the sample were 3.92 min and 11.46 min, respectively, which were not significantly different from cement without β-TCP. The degradation time of the BCPC material was longer than that of calcium phosphate cement alone. The healing process was significantly faster for BCPC than for the control and commercial product groups. Therefore, this is the first evidence that BCPC is an attractive option for bone surgery due to its faster stimulation of healing and faster degradation rate.

In situ production of pre-vascularized synthetic bone grafts for regenerating critical-sized defects in rabbits

Reconstructing large bone defects caused by severe trauma or resection of tumors remains a challenge for surgeons. A fibula free flap and its vascularized bed can be transplanted to the reconstruction site to achieve healing. However, this technique adds morbidity, and requires microsurgery and sculpting of the bone tissue to adapt the graft to both the vasculature and the anatomy of the defect. The aim of the current study was to evaluate an alternative approach consisting of the in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a critical-sized bone defect. 3D printed chambers containing biphasic calcium phosphate (BCP) granules, perfused by a local vascular pedicle, with or without the addition of stromal vascular fraction (SVF), were subcutaneously implanted into New Zealand White female rabbits. SVF was prepared extemporaneously from autologous adipose tissue, the vascular pedicle was isolated from the inguinal site, while BCP granules alone served as a control group. After 8 weeks, the constructs containing a vascular pedicle exhibited abundant neovascularization with blood vessels sprouting from the pedicle, leading to significantly increased vascularization compared to BCP controls. Pre-vascularized synthetic bone grafts were then transplanted into 15 mm critical-sized segmental ulnar defects for a further 8 weeks. Micro-CT and decalcified histology revealed that pre-vascularization of synthetic bone grafts led to enhanced bone regeneration. This pre-clinical study demonstrates the feasibility and efficacy of the in situ production of pre-vascularized synthetic bone grafts for regenerating large bone defects, thereby addressing an important clinical need. Statement of significanceThe current gold standard in large bone defect regeneration is vascularized fibula grafting. An alternative approach consisting of in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a bone defect is presented here. 3D printed chambers were filled with biphasic calcium phosphate granules, supplemented with autologous stromal vascular fraction and an axial vascular pedicle and subcutaneously implanted in inguinal sites. These pre-vascularized synthetic grafts were then transplanted into critical-sized segmental ulnar defects. Micro-CT and decalcified histology revealed that the pre-vascularized synthetic bone grafts led to higher bone regeneration than non-vascularized constructs. An alternative to vascularized fibula grafting is provided and may address an important clinical need for large bone defect reconstruction.

Functionalization of extracellular matrix (ECM) on multichannel biphasic calcium phosphate (BCP) granules for improved bone regeneration

Extracellular matrix (ECM) was functionalized onto the surface of multi-channel biphasic calcium phosphate granules (MCG) to enhance its osteogenic potential and bone regeneration capacity. ECM functionalized MCG scaffolds contains collagen, sulfated glycosaminoglycan and trace amounts of growth factors such as bone morphogenic protein 2 (BMP-2) vascular endothelial growth factor (VEGF), transforming growth factor (TGF) and fibroblast growth factor (FGF). ECM functionalized MCG supported proliferation of MC3T3-E1 cells after 7 days. Improved protein adsorption in ECM functionalized MCG scaffolds was observed after 15 min of incubation. Osteogenic properties were improved in ECM functionalized MCG scaffolds based on ALP and mineralization of MC3T3-E1 cells after 7 and 14 days. Increased bone regeneration was observed in ECM functionalized MCG scaffolds implanted in rabbit femoral head defect after 4 and 8 weeks. Results showed that ECM functionalized MCG scaffolds enhanced bone regeneration and could be a promising biomaterial in bone regeneration application.

Bioactivity of Biphasic Calcium Phosphate Granules, the Control of a Needle-Like Apatite Layer Formation for Further Medical Device Developments.

Biphasic calcium phosphate (BCP) bioceramics (hydroxyapatite/tricalcium phosphate, or HA/TCP) for tissue engineering and drug delivery systems is a unique know-how. A mechanical mixture of HA and TCP does not lead to such bioactive ceramics. The wet elaboration conditions of calcium-deficient apatite (CDA) or CDHA, followed by sintering, converts it into TCP and HA. The dissolution precipitation of nano-sized needle-like crystals at the surface of BCP occurs on time at body temperature. Combining several technics of characterization [scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive x-ray spectroscopy (EDX), Brunauer-Emmett-Teller method (BET), chemical analysis, x-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR)], we demonstrated an evolution on time of the HA/β-TCP. The current paper describes the crystallographic evolution of initial β-TCP rhombohedral crystallographic structure to microsized needle-like layer corresponding to apatitic TCP form. This phenomenon leads to an increase of the HA/TCP ratio, since hexagonal apatitic TCP is similar to hexagonal HA. However, the Ca/P ratio (reflecting the chemical composition HA/TCP) remains unchanged. Thus, the high reactivity of BCP involves dynamic evolution from rhombohedral to hexagonal structure, but not a chemical change. The dynamic process is reversible by calcination. These events are absolutely necessary for smart scaffolds in bone regeneration and orthobiology.

Implantation of an Injectable Bone Substitute Material Enables Integration Following the Principles of Guided Bone Regeneration.

The present study investigates the in vivo tissue reaction and the integration behavior of an injectable bone substitute material (IBS) composed of a water-based gel combined with nano hydroxyapatite particles and biphasic calcium phosphate granules. The results of the IBS were compared to biphasic bone substitute granules (BBSM) of the same chemical composition. The subcutaneous implantation model in 40 female 5-week-old CD-1 mice up to 60 days after implantation was used for conduction of the in vivo experiments. Moreover, established histological, histopathological and histomorphometrical methods were applied. The results showed that the IBS was gradually invaded by cells and complex tissue elements. Thus, the implant bed could be distinguished in two areas, i.e. an outer and inner region. While the outer region started to interact with the peri-implant tissue by evoking multinucleated giant cells and at earlier time points by undergoing a continuous high vascularization, the inner part was free of peri-implant cells for at least 30 days, starting to undergo a similar tissue reaction at a later time point. The bone substitute granules allowed for a fast tissue influx between the interspaces of the granules starting at day 10. While the vessel density did not differ in both groups up to the end of the study, the amount of vascularization was significantly higher over the entire observation period in the BBSM group. Moreover, the amount of biomaterial-associated multinucleated giant cells (BMGCs) was significantly higher in the IBS group in the period of between 15 to 30 days after implantation, while comparable BMGC numbers were found in both groups towards the end of the study. IBS can build a barrier-like structure that is able to control the soft tissue influx into the central regions of the implantation bed, which could not be observed in other bone substitute granules of the same chemical composition. This directed integration behavior is assumed to be in accordance with the concept of Guided Bone Regeneration (GBR). Furthermore, BMGCs can significantly influence the process of angiogenesis within an implant bed of a biomaterial but not the maturity of blood vessels.

&lt;i&gt;In Situ&lt;/i&gt; Production of Pre-Vascularized Synthetic Bone Grafts for Regenerating Critical-Sized Defects in Rabbits

Reconstructing large bone defects caused by severe trauma or resection of tumors remains a challenge for orthopedic and plastic surgeons. Free flaps comprised of a muscle flap with fibula bone and its vascularized bed can be transplanted to the reconstruction site to achieve healing. However, this transplantation technique adds morbidity, and requires extensive microsurgery and sculpting of the bone tissue to adapt the graft to both the vasculature and the anatomy of the bone defect. The aim of the current study is to evaluate an alternative approach consisting of the in situ production of a pre-vascularized synthetic bone graft and its subsequent transplantation to a critical-sized bone defect. 3D printed chambers containing biphasic calcium phosphate (BCP) granules, perfused by a local vascular pedicle, with or without the addition of stromal vascular fraction (SVF), were subcutaneously implanted into New Zealand White female rabbits. The SVF was prepared extemporaneously from autologous adipose tissue, while the vascular pedicle was isolated from the inguinal site. Chambers filled with BCP alone served as controls. After 8 weeks, the constructs containing a vascular pedicle exhibited abundant neovascularization, with significant numbers of blood vessels sprouting from the pedicle, which was further enhanced by the addition of SVF. These pre-vascularized synthetic bone grafts were then transplanted into 15 mm critical-sized segmental ulnar defects for a further 8 weeks. Micro-CT and decalcified histology revealed that pre-vascularization of synthetic bone grafts led to significantly enhanced bone regeneration. This pre-clinical study demonstrates the feasibility and efficacy of in situ production of pre-vascularized synthetic bone grafts for regenerating large bone defects, and will address an important clinical need.