Bone formation beyond the skeletal envelope using calcium phosphate granules packed into a collagen pouch-a pilot study.

In this proof-of-concept, bone neoformation beyond the skeletal envelope is explored by using a collagen pouch (n= 6) packed with calcium phosphate (CaP) granules placed over the frontal bone in sheep (n= 3). At 13 weeks, macroscopic examination showed specimens covered by an adherent fibrinous envelope with slight vascularization. Histology revealed colonization of the implant by newly formed woven bone and fibrous connective tissue. Surface osteoblasts as well as material-filled macrophages, lymphocytes, polymorphonuclear cells and giant cells were also found in large quantities surrounding the newly formed bone tissue inside the collagen pouch. On the side facing the recipient bone, the collagen membrane had to a large extent been resorbed and bridging bone formation was clearly visible between the test article and recipient bone. On the other side facing soft tissue, the collagen pouch remained intact with a visible fibrous capsule. This study demonstrated that the use of a collagen sleeve as a container for CaP granules allows for good neoformation beyond the skeletal envelope with bridging bone formation clearly visible between the test article and recipient bone. Additionally, in this model, macrophages rather than osteoclasts appear to modulate CaP granule resorption and remodeling into new bone. This construct opens new perspectives for treatment methods that could be used for bone augmentation and restoration of cranio-maxillofacial defects and malformations.

Bone without borders - Monetite-based calcium phosphate guides bone formation beyond the skeletal envelope.

Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (∼75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.

In situ bone regeneration of large cranial defects using synthetic ceramic implants with a tailored composition and design.

The repair of large cranial defects with bone is a major clinical challenge that necessitates novel materials and engineering solutions. Three-dimensionally (3D) printed bioceramic (BioCer) implants consisting of additively manufactured titanium frames enveloped with CaP BioCer or titanium control implants with similar designs were implanted in the ovine skull and at s.c. sites and retrieved after 12 and 3 mo, respectively. Samples were collected for morphological, ultrastructural, and compositional analyses using histology, electron microscopy, and Raman spectroscopy. Here, we show that BioCer implants provide osteoinductive and microarchitectural cues that promote in situ bone regeneration at locations distant from existing host bone, whereas bone regeneration with inert titanium implants was confined to ingrowth from the defect boundaries. The BioCer implant promoted bone regeneration at nonosseous sites, and bone bonding to the implant was demonstrated at the ultrastructural level. BioCer transformed to carbonated apatite in vivo, and the regenerated bone displayed a molecular composition indistinguishable from that of native bone. Proof-of-principle that this approach may represent a shift from mere reconstruction to in situ regeneration was provided by a retrieved human specimen, showing that the BioCer was transformed into well-vascularized osteonal bone, with a morphology, ultrastructure, and composition similar to those of native human skull bone.

Polyvinylalcohol-carbazate (PVAC) reduces red blood cell hemolysis.

BACKGROUND AND OBJECTIVES: The objective of this study was to investigate whether a soluble polymer and aldehyde-scavenger, polyvinylalcohol-carbazate (PVAC), can inhibit hemolysis in the storage of red blood cells (RBC). STUDY DESIGN AND METHODS: The effect of PVAC was assessed over a wide range of concentrations, using absorption spectroscopy to evaluate the level of hemolysis. Moreover, osmotic stability and aldehyde-scavenging potential of RBC were assessed after storage in PVAC. RESULTS: After test tube storage for two weeks, red blood cell hemolysis was lower with PVAC compared to controls (mean difference 23%, 95% CI 16-29%, p < 0.001). A higher level of hemolysis led to a pronounced effect with PVAC. RBC stored in PVAC improved both the binding of free aldehydes (p <0.001) and the osmotic stability (p = 0.0036). CONCLUSION: Erythrocytes stored with PVAC showed less hemolysis, which might be explained by the ability of PVACs to stabilize the cell membrane and decrease oxidative injury.

Patient-Specific Titanium-Reinforced Calcium Phosphate Implant for the Repair and Healing of Complex Cranial Defects.

BACKGROUND: The reconstruction of complex cranial defects is challenging and is associated with a high complication rate. The development of a patient-specific, titanium-reinforced, calcium phosphate-based (CaP-Ti) implant with bone regenerative properties has previously been described in 2 case studies with the hypothesis that the implant may improve clinical outcome. OBJECTIVE: To identify whether the introduction of CaP-Ti implant has the potential to improve clinical outcome. METHODS: A retrospective review of all patients having undergone CaP-Ti cranioplasty was conducted. Comprehensive clinical data were collected from the hospital computer database and patient records. Bone formation and osseointegration were analyzed in a single retrieval specimen. RESULTS: Fifty patients, with 52 cranial defects, met the inclusion criteria. The patient cohort displayed a previous failure rate of 64% (32/50) with autologous bone, alloplastic materials, or both. At a median follow-up time of 25 months, the explantation rate due to either early postoperative infection or persistent wound dehiscence was 1.9% (1/53) or 3.8% (2/53), respectively. Surgical intervention with local wound revision was required in 2 patients without the need of implant removal. One patient had a brain tumor recurrence, and the implant was explanted 31 months after implantation. Histologic examination showed that the entire implant was partly yet evenly transformed into vascularized compact bone. CONCLUSION: In the present study the CaP-Ti implant appears to have improved the clinical outcomes in a cohort of patients with a high rate of previous cranioplasty failures. The bone regenerative effect may in particular have an impact on the long-term success rate of the implant.

Pyrophosphate Stimulates Differentiation, Matrix Gene Expression and Alkaline Phosphatase Activity in Osteoblasts.

Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100µM and 0.1µM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100µM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100µM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression.

Bioceramic Implant Induces Bone Healing of Cranial Defects.

Autologous bone or inert alloplastic materials used in cranial reconstructions are techniques that are associated with resorption, infection, and implant exposure. As an alternative, a calcium phosphate-based implant was developed and previously shown to potentially stimulate bone growth. We here uncover evidence of induced bone formation in 2 patients. Histological examination 9 months postoperatively showed multinuclear cells in the central defect zone and bone ingrowth in the bone-implant border zone. An increased expression of bone-associated markers was detected. The other patient was investigated 50 months postoperatively. Histological examination revealed ceramic materials covered by vascularized compact bone. The bone regenerative effect induced by the implant may potentially improve long-term clinical outcome compared with conventional techniques, which needs to be verified in a clinical study.

Development of a bioactive implant for repair and potential healing of cranial defects.

The repair of complex craniofacial bone defects is challenging and a successful result is dependent on the size of the defect, quality of the soft tissue covering the defect, and choice of reconstruction method. The objective of this study was to develop a bioactive cranial implant that could provide a permanent reconstructive solution to the patient by stimulating bone healing of the defect. In this paper the authors report on the feasibility and clinical results of using such a newly developed device for the repair of a large traumatic and therapy-resistant cranial bone defect. The patient had undergone numerous attempts at repair, in which established methods had been tried without success. A mosaic-designed device was manufactured and implanted, comprising interconnected ceramic tiles with a defined calcium phosphate composition. The clinical outcome 30 months after surgery revealed a restored cranial vault without postoperative complications. Computed tomography demonstrated signs of bone ingrowth. Examination with combined (18)F-fluoride PET and CT provided further evidence of bone healing of the cranial defect.

Variation in calvarial bone healing capacity: a clinical study on the effects of BMP-2-hydrogel or bone autograft treatments at different cranial locations.

BACKGROUND: Bone morphogenetic protein-2 (BMP-2) together with a suitable carrier is an attractive option that may be used for craniofacial bone reconstruction. In this prospective randomized study, a hyaluronan-based hydrogel with BMP-2 was used to achieve bone healing in standardized critical-size cranial defects in humans after neurosurgery. METHODS: Twelve patients were randomized into the treatment group (N = 6) or control group (N = 6). In the treatment group, holes made during craniotomy were treated with hydrogel with BMP-2, 250 µg/mL, or hydrogel without BMP-2. In the remaining hole/s in the same patient, Spongostan (Ethicon) alone or Tisseel (Baxter) mixed with autologous bone matrix were used as negative and positive controls, respectively. In the control group, the holes were treated with Spongostan or Tisseel mixed with bone autograft. Bone healing was assessed with CT scans after 3 and 6 months. Bone areas in treated defects were measured and statistical analysis was performed. RESULTS: Independent of location, bone healing in defects treated with Tisseel with autograft, hydrogel alone, or hydrogel with BMP-2 was significantly increased compared to negative control (P < 0.001, P = 0.002, and P = 0.005, respectively). In general, all defects healed significantly better in the frontal bone as compared to parietal-temporal location, except for defects treated with Tisseel and autograft, which healed well independently of location. No local or systemic side effects, including excessive bone overgrowth or inflammatory reaction, were seen in treated patients. CONCLUSIONS: Tissue engineering of bone with hyaluronan-based hydrogel shows good healing of cranial defects, comparable with bone autografts. The hydrogel itself may represent a novel alternative to autologous bone transplants in craniofacial bone repair. The study also reveals a general superior healing capacity in the frontal bone as compared to parietal/temporal bones.

Minimally invasive mandibular bone augmentation using injectable hydrogels.

Hyaluronic acid-based hydrogels are proven biocompatible materials and excellent carriers of bone morphogenetic protein-2 (BMP-2) that have been successfully tested for bone generation in vivo. Different formulations, with or without nanohydroxyapatite, have shown promise for craniofacial applications. In this study, 28 rats were used to investigate whether it is possible to achieve mandibular bone augmentation upon injection of novel hyaluronic acid-based hydrogels containing nanohydroxyapatite and different concentrations of BMP-2 (0, 5 and 150 µg/ml). The biomaterials were injected subperiosteally through fine needles into the innate mandibular diastema, imitating a clinical procedure for resorbed mandibles. No incisions, flaps or sutures were necessary. After 8 weeks the mandibles were evaluated by peripheral quantitative computed tomography (pQCT), micro-computed tomography (µCT), histology, immunohistochemistry and fluorochrome labelling. As a result, engineered bone was observed in all treated mandibles, with a statistically significant increase in mandibular bone volume correlated with the amount of BMP-2 loaded in the hydrogel formula. We therefore demonstrated that minimally invasive mandibular bone augmentation is possible upon injection in rats, when using the appropriate injectable scaffolds. This represents an attractive clinical alternative for oral implantology patients.

Biomaterials and biologics in craniofacial reconstruction.

Complications related to surgery, including infection, wound dehiscence, and implant protrusion, are costly and may cause severe morbidity to patients. The choice of implants materials is critical for a successful outcome, particularly in craniofacial reconstructions. This review discusses the potential benefits and drawbacks of biologically active materials used for craniofacial bone repair as alternatives to inert implant prostheses.

In situ cross-linkable hyaluronan hydrogel enhances chondrogenesis.

The present work describes the feasibility of a cross-linkable injectable hyaluronan hydrogel for cartilage repair. The hydrogel used is a two-component system based on aldehyde-modified hyaluronan and hydrazide-modified polyvinyl alcohol, which are rapidly cross-linked in situ upon mixing. The in vitro study showed that chondrocytes and mesenchymal cells cultured in the gel form cartilage-like tissue, rich in glycosaminoglycans, collagen type II and aggrecan. In a rabbit animal model the injection of the hydrogel improved the healing of a full-thickness cartilage defect created in the knee as compared to non-treated controls. This rabbit study showed that the regenerated cartilage defects stained more intensely for type II collagen upon treatment with the hydrogel. The hyaluronan-based hydrogel may be used as a delivery vehicle for both growth factors and/or cells for cartilage repair. The in vivo study also indicated that the hydrogel alone has a beneficial effect on cartilage regeneration.

Bone morphogenetic protein-2 delivered by hyaluronan-based hydrogel induces massive bone formation and healing of cranial defects in minipigs.

BACKGROUND: Reconstruction of large craniofacial bone defects is a challenge using bone transplants or alloplastic materials. The use of bone morphogenetic protein (BMP)-2 together with a suitable carrier is an attractive option that may facilitate new bone formation. The authors have developed a hydrogel that is formed in situ by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives mixed with hydroxyapatite nanoparticles, in the presence of BMP-2. The aim of this study was to evaluate the suitability of the hydrogel as a carrier for BMP-2 in repairing critical size cranial defects in a minipig model. METHODS: Cranial defects (2 x 4 cm) were created in 14 minipigs. The experimental groups were as follows: group 1, craniotomy and application of 5 ml of hydrogel with 1.25 mg of BMP-2 (n = 6); group 2, craniotomy and application of 5 ml of hydrogel without BMP-2 (n = 6); and group 3, craniotomy with no further treatment (n = 2). RESULTS: After 3 months, computed tomographic and histologic examinations were performed. There was spontaneous ossification in the untreated group, but the healing was incomplete. The hydrogel alone demonstrated no further effects. The addition of 1.25 mg of BMP-2 to the hydrogel induced a greater than 100 percent increase in bone volume (p = 0.003) and complete healing of the defects. Histologic examination revealed compact lamellar bone in the BMP group without intertrabecular fibrous tissue, as was seen in the other groups. The hydrogel was resorbed completely within 3 months and, importantly, caused no inflammatory reaction. CONCLUSION: The injectable hydrogel may be favorable as a BMP-2 carrier for bone reconstruction.

Craniofacial reconstruction with bone and biomaterials: review over the last 11 years.

This review aims to compare bone grafts and different biomaterials for reconstruction of craniofacial bones in congenital defects, after trauma, and after tumour surgery. A Pubmed search was performed and publications over the last 11 years describing reconstructions of craniofacial bones in non-load-bearing areas were reviewed. Only human studies using bone grafts and biomaterials were included. Studies on skull base reconstruction, distraction osteogenesis, free and pedicled bone flaps and bone-anchored epithesis were excluded. Out of 83 studies, three were prospective, 65 retrospective and 15 studies were case reports. There were seven comparative studies found and some efforts on statistical analysis were made. Except for a few studies, the statistical significant differences in outcomes were found to be related to size and location of bone defects rather than reconstruction method and biomaterial used. An increasing number of alloplastic materials have been available as alternatives to the gold standard autologous bone transplantation for craniofacial bone repair. Comparative studies with statistical analyses on differences in success rates between different biomaterials or bone grafts for specific indications are needed.

Injectable cell-free template for bone-tissue formation.

Here we present a novel injectable hydrogel which forms a template for de novo formation of bone tissue. Hydrogel formation takes place in situ in less than 1 min by the cross-linking of multifunctional hyaluronic acid and polyvinyl alcohol derivatives. Endogenous cells are recruited in vivo by incorporating bone morphogenetic protein-2 (BMP-2), a powerful promoter for osteogenic differentiation. The hydrogel was evaluated in vitro by performing a cell viability test and a release study and in vivo by a rat ectopic model. Examination by X-ray, microcomputed tomography, and histology revealed a significant bone formation at the target site for gels containing BMP-2, and a complete degradation was observed for gels without BMP-2 four weeks after injection. There were no signs of inflammation or foreign body response in either group and we believe that this system has the potential as an off-the-shelf injectable to be used where bone tissue is needed.

A novel biodegradable delivery system for bone morphogenetic protein-2.

BACKGROUND: The efficacy of recombinant growth factors in vivo is highly dependent on the delivery vehicle. The authors investigated the osteoinductive effects of recombinant human bone morphogenetic proteins (BMP)-2 implanted together with a complex of heparin and chitosan. METHODS: Sixty rats were used. Three different carriers in gel formulation (type I collagen, heparin/type I collagen, and heparin/chitosan) were mixed with either 0, 10, or 50 microg of BMP-2, making the number of groups nine. The gels were injected into the quadriceps muscles of both legs in 45 rats (n = 10 per group). Freeze-dried formulations of the carriers were also tested with the same amounts of BMP-2 using 15 rats (n = 5 per group). Four weeks after implantation, the quality and amount of newly formed bone were assessed. RESULTS: Chitosan was shown to protect the heparinase-mediated degradation of heparin in vitro. The osteoinductive effects of BMP-2 in combination with heparin/chitosan were superior as compared with BMP-2 implanted together with type I collagen. Interestingly, the heparin/chitosan complex induced a small amount of bone also without BMP-2 added. The heparin/chitosan was completely absorbed after 4 weeks as determined by histologic evaluation, and a normal active bone formation was present. The freeze-dried formulations of the carriers demonstrated similar osteoinductive effects as the gels. CONCLUSIONS: An osteoinductive formula for clinical use is needed for general bone reconstruction. Heparin in complex with chitosan has the ability to stabilize or activate the growth factor in vivo and induce the generation of new bone in good yields.

Three-dimensional technology and bone morphogenetic protein in frontal bone reconstruction.

Osteoinductive bone morphogenetic proteins (BMPs) may be used in humans to facilitate healing of bony defects. The effect of different BMPs is, as with many other growth factors, highly dependent on the delivery vehicle. Bovine type I collagen is currently used in the clinical setting as a carrier and has been approved in several countries for human use. Here, we report the reconstruction of a frontal bone defect using heparin together with bovine type I collagen, hyaluronic acid, and fibrin as vehicles for BMP-2. A bony structure was created on the back of the patient by treating the latissimus dorsi muscle with the growth factor. A polyamide mold was used as a template to achieve the desired shape. The bone structure was transplanted into the defect site via microsurgical techniques. Although the prefabricated bone was not large enough tocover the entire frontal defect, the reconstruction was completed by using an additional cranial implant.

Regeneration of human auditory nerve. In vitro/in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion.

Time lapse video recordings of cultured adult human and guinea pig spiral ganglion (hSG and gpSG) show that mitogen responsive progenitor/stem cells develop in the form of spheres that proliferate and differentiate into mature neurons and glia cells. Neurospheres, cultured with EGF and bFGF showed expression of nestin and incorporation of 5'-Bromo-2-deoxyuridine (BrdU). Newly formed BrdU labelled cells were positive for beta-tubulin, and also for GFAP demonstrating that neuronal cells were derived from a dividing population of progenitor cells. Dissociated spheres cultured either with glia cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), induced differentiation of the progenitor cells. Video microscopy showed that neurons develop from subcultured spheres maintained for up to four weeks. Neurons showed fasciculation and migration with a speed of 10-30 microm/h, and some cells had up to 6 mm long neurites coexpressing TrkB and TrkC receptors. Precise dissection suggests that the neurons formed are cochlea-specific. The results suggest that the mammalian auditory nerve has the capability for self-renewal and replacement. Transplantation of progenitor cells together with established means to induce neural differentiation and fiber growth may facilitate strategies for better repair and treatment of auditory neuronal damage.

Molecular biologic aspects of cartilage and bone: potential clinical applications.

The formation of cartilage and bone tissue from condensing mesenchymal stem cells is taking place in the early stage embryo, but also in the growth plate and during fracture repair in adults. Resident mesenchymal stem cells have been identified in bone marrow, periosteum, and in muscles. These pluripotent cells are attractive as therapeutic cells in cartilage and bone reconstructive procedures, since they can differentiate into chondrocytes and osteoblasts upon appropriate stimuli, such as certain growth factors. Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta1-3, bone morphogenetic proteins (BMPs), and activins, are essential mediators in cell proliferation and differentiation and play regulatory roles in cartilage and bone formation. This review presents functional data on TGF-beta1 in cartilage formation, BMP2 and BMP3 in bone formation, and a role of the BMP-regulatory protein noggin in BMP2 processing. Potential clinical applications of these proteins are further demonstrated and discussed.