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 SIGNIFICANCE: The 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.

Vertical Bone Regeneration with Synthetic Biomimetic Calcium Phosphate onto the Calvaria of Rats.

IMPACT STATEMENT: This work reports a new bone substitute made of precipitated apatite crystals that resemble in composition and crystallinity to the mineral phase of bone. The bone regeneration capacity of this synthetic biomimetic calcium phosphate (SBCP) was studied by using an original model of vertical bone regeneration with cups on the calvaria of rats. After 4 weeks, a significantly higher bone growth was found with SBCP compared with deproteinized bovine bone matrix and empty controls. This rapid vertical bone regeneration indicated that this new biomaterial is particularly interesting for filling bone defects in oral surgery.

Combination of blood and biphasic calcium phosphate microparticles for the reconstruction of large bone defects in dog: A pilot study.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.

Characterization of the age-dependent intervertebral disc changes in rabbit by correlation between MRI, histology and gene expression.

BACKGROUND: The present study was conducted to address whether the intervertebral disc of rabbit could be considered (i) as a valuable model to provide new insights into the tissue and cellular changes of Nucleus pulposus aging and (ii) as an appropriate tool to investigate the efficacy of Nucleus pulposus cell-based biotherapies. METHODS: Lumbar intervertebral disc from rabbits with increasing ages (1, 6 and 30 month-old) were compared by MRI and histological observation using Pfirrmann's grading and Boos' scoring respectively. The expression of transcripts (COL2A1, AGC1, COL1A1, MMP13, BMP2, MGP and p21) in Nucleus pulposus cells were analysed by quantitative real-time PCR. RESULTS: MRI analysis indicated an early age-dependent increase in the Pfirrmann's grading. Histological Boos' scoring was also increased. The analysis of transcript expression levels showed that COL2A1 and AGC1 were down-regulated as a function of age. Conversely, COL1A1, MMP-13, BMP-2, MGP and p21 were significantly up-regulated in the Nucleus pulposus cells of aged rabbit intervertebral disc. CONCLUSIONS: Our study describes the consistency of the rabbit as a model of intervertebral disc changes as a function of age by correlating tissue alteration with cellular modification measured.

Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate.

An injectable composite silanized hydroxypropyl methyl cellulose/biphasic calcium phosphate (Si-HPMC/BCP) has been investigated in humans with promising results. The aim of this study was to evaluate his efficacy for treating periodontal defects (canine fenestration and premolar furcation) in dog models. At 3 months, we observed that bone formation around BCP particles in furcation model is more discernible but not statistically significant in defects filled with Si-HPMC/BCP compared to healing in control. We suggest that BCP particles sustain the bone healing process by osteoconduction, while the Si-HPMC hydrogel enhances intergranular cohesion and acts as an exclusion barrier. Furthermore, bone ingrowth is not so distinctive in superficial defects where the biomaterial appears unstable. These results with Si-HPMC/BCP are encouraging. In addition, this biomaterial is easy to use and simplifies the process of filling periodontal lesions. However, more researches are needed to improve the viscosity and hardness to adjust the material to the specificities of periodontal defects.

The in vivo degradation of a ruthenium labelled polysaccharide-based hydrogel for bone tissue engineering.

In this paper we report a new method that permitted for the first time to selectively track a polysaccharide-based hydrogel on bone tissue explants, several weeks after its implantation. The hydrogel, which was developed for bone healing and tissue engineering, was labelled with a ruthenium complex and implanted into rabbit bone defects in order to investigate its in vivo degradation. 1, 2, 3 and 8 weeks after surgery, the bone explants were analyzed by synchrotron X-ray microfluorescence, infrared mapping spectroscopy, scanning electron microscopy, and optical microscopy after histological coloration. The results showed that the labelled polysaccharide-based hydrogel was likely to undergo phagocytosis that seemed to occur from the edge to the center of the implantation site up to at least the 8th week.

Sol-gel synthesis and characterization of macroporous calcium phosphate bioceramics containing microporosity.

Amorphous calcium phosphate powders were precipitated from calcium metal and phosphoric acid in ethanol. Depending on the quantity of reagent, the CaP powders had different chemical compositions and, after heating, formed beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) or BCP mixtures. Dilatometric measurements indicated that shrinkage of compacted CaP powders occurred first at around 650 degrees C and continued up to 1200 degrees C. The amorphous CaP powders were mixed with urea beads, compacted under isostatic pressure at 140 MPa and sintered at 1100 degrees C for 5 h. Scanning electron microscopy indicated that macro-microporous ceramics were produced. The ceramics had spherical macropores of 700-1200 microm in diameter, with limited interconnections and a macroporosity of 42% as determined by microcomputed tomography. The micropores ranged from 0.1 to 1 microm in diameter. These ceramics made of HA, beta-TCP or BCP exhibiting both macroporosity and microporosity can be used as bone fillers.

Osteogenicity of biphasic calcium phosphate ceramics and bone autograft in a goat model.

The aim of this work was to compare the osteogenicity of calcium phosphate ceramic granules with autologous bone graft in ectopic and orthotopic sites. Biphasic calcium phosphate (BCP) granules composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in a 60/40 ratio were sintered at 1050, 1125 and 1200 degrees C, producing different microporosities. Either BCP ceramic granules or autologous bone chips (n=7) were implanted into paraspinal muscles. Osteoinduction was not observed in either the BCP implants or autologous bone chips after 6 or 12 weeks in the ectopic sites. Hollow and bored polytetrafluoroethylene (PTFE) cylinders were filled with autologous bone, BCP granules or left empty, then implanted into critical-sized defects in femoral epiphyses. The PTFE cylinders left empty contained marrow and blood vessels but not mineralized bone, indicating that this model prevented bone ingrowth (0.56+/-0.43% at 12 weeks). Bone formation was observed in contact with the BCP1050 and BCP1125 granules in the femoral sites after 6 weeks. The amount of bone after 12 weeks was 5.6+/-7.3 and 9.6+/-6.6% for BCP1050 and BCP1125, respectively. Very little bone formation was observed with the BCP1200 implants (1.5+/-1.3% at 12 weeks). In both the ectopic and orthotopic sites, autologous bone chips were drastically resorbed (from 19.4+/-3.7% initially to 1.7+/-1.2% at 12 weeks). This study shows that synthetic bone substitutes may have superior stability and osteogenic properties than autologous bone grafts in critical-sized bone defects.

Inflammatory reaction in rats muscle after implantation of biphasic calcium phosphate micro particles.

Several studies have shown that macro micro porous bioceramics ectopically implanted promote bone tissue formation. This study aims at investigating the inflammatory response towards biphasic calcium phosphate (BCP) ceramic micro particles. BCP composed of hydroxyapatite (HA) and beta-tricalcium phosphate, HA/beta -TCP ratio of 50/50, were prepared by sintering at 1200 degrees C for 5 h. After crushing, 3 fractions of BCP micro particles < 20, 40-80 and 80-200 micro m were sieved. The micro particles were carefully characterized by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and laser scattering. The inflammatory reactions induced by BCP micro particles implanted in quadriceps muscles of rats for 7, 14 and 21 days were studied by histology (n = 8/group). A fibrous tissue encapsulation of the BCP micro particles implanted in muscle tissue was observed and fibrosis was similar for the 3 groups of micro particles. The comparison of the cellular response indicated that the total number of cells was significantly higher for BCP < 20 micro m than for 40-80 and 80-200 micro m (p < 0.0001). The number of macrophages was relatively higher for the smallest than for the intermediate and largest fractions (p < 0.0001). The relative percentage of giant cells was higher for the intermediate and largest size of particles than for the smallest. The number of lymphocytes was comparable for the 3 fractions and after the 3 delays. Therefore, the BCP micro particles < 20 micro m initiated an inflammatory response which might play an important role in osteogenesis.

Bone repair using a new injectable self-crosslinkable bone substitute.

A new injectable and self-crosslinkable bone substitute (IBS2) was developed for filling bone defects. The IBS2 consisted of a chemically modified polymer solution mixed with biphasic calcium phosphate (BCP) ceramic particles. The polymer hydroxypropylmethyl cellulose was functionalized with silanol groups (Si-HPMC) and formed a viscous solution (3 wt %) in alkaline medium. With a decrease in pH, self-hardening occurred due to the formation of intermolecular -Si-O- bonds. During setting, BCP particles, 40 to 80 microm in diameter, were added to the polymer solution at a weight ratio of 50/50. The resulting injectable material was bilaterally implanted into critically sized bone defects at the distal femoral epiphyses of nine New Zealand White rabbits. The IBS2 filled the bone defects entirely and remained in place. After 8 weeks, bone had grown centripetally and progressed towards the center of the defects. Newly formed bone, ceramic, and nonmineralized tissue ratios were 24.6% +/- 5.6%, 21.6% +/- 5.8%, and 53.7% +/- 0.1%, respectively. Mineralized and mature bone was observed between and in contact with the BCP particles. The bone/ceramic apposition was 73.4% +/- 10.6%. The yield strength for the IBS2-filled defects was 16.4 +/- 7.2 MPa, significantly higher than for the host trabecular bone tissue (2.7 +/- 0.4 MPa). This study showed that BCP particles supported the bone healing process by osteoconduction while the Si-HPMC hydrogel created intergranular space for bone ingrowth. This new injectable and self-crosslinkable bone substitute could be used conveniently in orthopedic surgery for filling critical-size bone defects.