From benchtop to clinic: a translational analysis of the immune response to submicron topography and its relevance to bone healing.

Proper regulation of the innate immune response to bone biomaterials after implantation is pivotal for successful bone healing. Pro-inflammatory M1 and anti-inflammatory M2 macrophages are known to have an important role in regulating the healing response to biomaterials. Materials with defined structural and topographical features have recently been found to favourably modulate the innate immune response, leading to improved healing outcomes. Calcium phosphate bone grafts with submicron-sized needle-shaped surface features have been shown to trigger a pro-healing response through upregulation of M2 polarised macrophages, leading to accelerated and enhanced bone regeneration. The present review describes the recent research on these and other materials, all the way from benchtop to the clinic, including in vitro and in vivo fundamental studies, evaluation in clinically relevant spinal fusion models and clinical validation in a case series of 77 patients with posterolateral and/or interbody fusion in the lumbar and cervical spine. This research demonstrates the feasibility of enhancing biomaterial-directed bone formation by modulating the innate immune response through topographic surface features.

Accelerated bone formation by biphasic calcium phosphate with a novel sub-micron surface topography.

Osteoinductive calcium phosphate (CaP) bone grafts have equivalent performance to autografts in repairing critical-size bone defects. The osteoinductive potential of CaP is linked to the size of the surface topographical features. In the present study, two novel biphasic calcium phosphate (BCP) bone grafts were synthesised with either sub-micron- (BCP<µm) or micron-scale (BCPµm) needle-shaped surface topography and compared to dimensionally similar tricalcium phosphate (TCP) with grain-shaped surface structures (TCP<µm and TCPµm). To clarify the possible function of the surface morphology (needle-like vs. grain-like) in initiating bone formation, the four CaP test materials were physicochemically characterised and implanted for 12 weeks in the dorsal muscle of beagles. The sub-micron needle-shaped topography of BCP<µm triggered earlier bone formation (3-6 weeks) as compared to the grain-shaped surface topography of TCP<µm, which formed bone at 6-9 weeks. After 12 weeks, the amount of induced bone formation in both materials was equivalent, based on histomorphometry. The micron-sized needle-shaped surface topography of BCPµm led to limited formation of new bone tissue, whereas its counterpart, TCPµm with grain-shaped surface topography, failed to trigger de novo bone formation. The relative strength of the parameters affecting CaP-driven bone induction was as follows: surface feature size > surface feature morphology > substrate chemistry. BCP materials with needle-shaped sub-micron surface topography gave rise to accelerated bone formation and slower rate of resorption than a comparable TCP. These characteristics may be translated to improve bone healing in orthotopic defects.

Influence of surface microstructure and chemistry on osteoinduction and osteoclastogenesis by biphasic calcium phosphate discs.

It has been reported that surface microstructural dimensions can influence the osteoinductivity of calcium phosphates (CaPs), and osteoclasts may play a role in this process. We hypothesised that surface structural dimensions of ≤ 1 µm trigger osteoinduction and osteoclast formation irrespective of macrostructure (e.g., concavities, interconnected macropores, interparticle space) or surface chemistry. To test this, planar discs made of biphasic calcium phosphate (BCP: 80% hydroxyapatite, 20% tricalcium phosphate) were prepared with different surface structural dimensions - either ~ 1 µm (BCP1150) or ~ 2-4 µm (BCP1300) - and no macropores or concavities. A third material was made by sputter coating BCP1150 with titanium (BCP1150Ti), thereby changing its surface chemistry but preserving its surface structure and chemical reactivity. After intramuscular implantation in 5 dogs for 12 weeks, BCP1150 formed ectopic bone in 4 out of 5 samples, BCP1150Ti formed ectopic bone in 3 out of 5 samples, and BCP1300 formed no ectopic bone in any of the 5 samples. In vivo, large multinucleated osteoclast-like cells densely colonised BCP1150, smaller osteoclast-like cells formed on BCP1150Ti, and osteoclast-like cells scarcely formed on BCP1300. In vitro, RAW264.7 cells cultured on the surface of BCP1150 and BCP1150Ti in the presence of osteoclast differentiation factor RANKL (receptor activator for NF-κB ligand) proliferated then differentiated into multinucleated osteoclast-like cells with positive tartrate resistant acid phosphatase (TRAP) activity. However, cell proliferation, fusion, and TRAP activity were all significantly inhibited on BCP1300. These results indicate that of the material parameters tested - namely, surface microstructure, macrostructure, and surface chemistry - microstructural dimensions are critical in promoting osteoclastogenesis and triggering ectopic bone formation.

Submicron-scale surface architecture of tricalcium phosphate directs osteogenesis in vitro and in vivo.

A current challenge of synthetic bone graft substitute design is to induce bone formation at a similar rate to its biological resorption, matching bone's intrinsic osteoinductivity and capacity for remodelling. We hypothesise that both osteoinduction and resorption can be achieved by altering surface microstructure of beta-tricalcium phosphate (TCP). To test this, two TCP ceramics are engineered with equivalent chemistry and macrostructure but with either submicron- or micron-scale surface architecture. In vitro, submicron-scale surface architecture differentiates larger, more active osteoclasts--a cell type shown to be important for both TCP resorption and osteogenesis--and enhances their secretion of osteogenic factors to induce osteoblast differentiation of human mesenchymal stem cells. In an intramuscular model, submicrostructured TCP forms 20 % bone in the free space, is resorbed by 24 %, and is densely populated by multinucleated osteoclast-like cells after 12 weeks; however, TCP with micron-scale surface architecture forms no bone, is essentially not resorbed, and contains scarce osteoclast-like cells. Thus, a novel submicron-structured TCP induces substantial bone formation and is resorbed at an equivalent rate, potentially through the control of osteoclast-like cells.

Poly(trimethylene carbonate) and biphasic calcium phosphate composites for orbital floor reconstruction: a feasibility study in sheep.

In the treatment of orbital floor fractures, bone is ideally regenerated. The materials currently used for orbital floor reconstruction do not lead to the regeneration of bone. Our objective was to render polymeric materials based on poly(trimethylene carbonate) (PTMC) osteoinductive, and to evaluate their suitability for use in orbital floor reconstruction. For this purpose, osteoinductive biphasic calcium phosphate (BCP) particles were introduced into a polymeric PTMC matrix. Composite sheets containing 50 wt% BCP particles were prepared. Also laminates with poly(D,L-lactide) (PDLLA) were prepared by compression moulding PDLLA films onto the composite sheets. After sterilisation by gamma irradiation, the sheets were used to reconstruct surgically-created orbital floor defects in sheep. The bone inducing potential of the different implants was assessed upon intramuscular implantation. The performance of the implants in orbital floor reconstruction was assessed by cone beam computed tomography (CBCT). Histological evaluation revealed that in the orbital and intramuscular implantations of BCP containing specimens, bone formation could be seen after 3 and 9 months. Analysis of the CBCT scans showed that the composite PTMC sheets and the laminated composite sheets performed well in orbital floor reconstruction. It is concluded that PTMC/BCP composites and PTMC/BCP composites laminated with PDLLA have osteoinductive properties and seem suitable for use in orbital floor reconstruction.

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique.

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.

Heterotopic bone formation by nano-apatite containing poly(D,L-lactide) composites.

To render polymeric materials osteoinductive, nano-sized calcium phosphate apatite particles (CaP) were introduced into a low molecular weight poly(D,L-lactide). Homogenous composites were made with 10%, 20% and 40% by weight of apatite content while pure polylactide was used as control. Thereafter porous samples (pore size 300-400 microm, 60% porosity) were fabricated and sterilized. In vitro studies showed that calcium ions were released from the composites depending on the apatite content, while surface mineral deposition was observed only on the 40% CaP composites in simulated body fluid (SBF) within 14 days. After 12 weeks of intramuscular implantation in dogs, only the 40% CaP composite implant retained its shape and showed ectopic bone formation within the pores. In conclusion, adding a content of 40% apatite into poly(D,L-lactide) could lead to an osteoinductive material. Future studies will focus on understanding this phenomenon of material-directed osteoinduction in order to develop a promising bone graft substitute.

Ectopic bone formation in bone marrow stem cell seeded calcium phosphate scaffolds as compared to autograft and (cell seeded) allograft.

Improvements to current therapeutic strategies are needed for the treatment of skeletal defects. Bone tissue engineering offers potential advantages to these strategies. In this study, ectopic bone formation in a range of scaffolds was assessed. Vital autograft and devitalised allograft served as controls and the experimental groups comprised autologous bone marrow derived stem cell seeded allograft, biphasic calcium phosphate (BCP) and tricalcium phosphate (TCP), respectively. All implants were implanted in the back muscle of adult Dutch milk goats for 12 weeks. Micro-computed tomography (microCT) analysis and histomorphometry was performed to evaluate and quantify ectopic bone formation. In good agreement, both microCT and histomorphometric analysis demonstrated a significant increase in bone formation by cell-seeded calcium phosphate scaffolds as compared to the autograft, allograft and cell-seeded allograft implants. An extensive resorption of the autograft, allograft and cell-seeded allograft implants was observed by histology and confirmed by histomorphometry. Cell-seeded TCP implants also showed distinct signs of degradation with histomorphometry and microCT , while the degradation of the cell-seeded BCP implants was negligible. These results indicate that cell-seeded calcium phosphate scaffolds are superior to autograft, allograft or cell-seeded allograft in terms of bone formation at ectopic implantation sites. In addition, the usefulness of microCT for the efficient and non-destructive analysis of mineralised bone and calcium phosphate scaffold was demonstrated.

Surface-enrichment with hydroxyapatite nanoparticles in stereolithography-fabricated composite polymer scaffolds promotes bone repair.

Fabrication of composite scaffolds using stereolithography (SLA) for bone tissue engineering has shown great promises. However, in order to trigger effective bone formation and implant integration, exogenous growth factors are commonly combined to scaffold materials. In this study, we fabricated biodegradable composite scaffolds using SLA and endowed them with osteopromotive properties in the absence of biologics. First we prepared photo-crosslinkable poly(trimethylene carbonate) (PTMC) resins containing 20 and 40wt% of hydroxyapatite (HA) nanoparticles and fabricated scaffolds with controlled macro-architecture. Then, we conducted experiments to investigate how the incorporation of HA in photo-crosslinked PTMC matrices improved human bone marrow stem cells osteogenic differentiation in vitro and kinetic of bone healing in vivo. We observed that bone regeneration was significantly improved using composite scaffolds containing as low as 20wt% of HA, along with difference in terms of osteogenesis and degree of implant osseointegration. Further investigations revealed that SLA process was responsible for the formation of a rich microscale layer of HA corralling scaffolds. To summarize, this work is of substantial importance as it shows how the fabrication of hierarchical biomaterials via surface-enrichment of functional HA nanoparticles in composite polymer stereolithographic structures could impact in vitro and in vivo osteogenesis. STATEMENT OF SIGNIFICANCE: This study reports for the first time the enhance osteopromotion of composite biomaterials, with controlled macro-architecture and microscale distribution of hydroxyapatite particles, manufactured by stereolithography. In this process, the hydroxyapatite particles are not only embedded into an erodible polymer matrix, as reported so far in the literature, but concentrated at the surface of the structures. This leads to robust in vivo bone formation at low concentration of hydroxyapatite. The reported 3D self-corralling composite architecture provides significant opportunities to develop functional biomaterials for bone repair and tissue engineering.

A comparison of bone formation in biphasic calcium phosphate (BCP) and hydroxyapatite (HA) implanted in muscle and bone of dogs at different time periods.

Physicochemical modification could implement synthetic materials into osteoinductive materials, which induce bone formation in nonosseous tissues. We hereby studied the relevance between the osteogenic capacities of osteoinductive materials in nonosseous tissues and in osseous sites. Biphasic calcium phosphate ceramic (BCP) and hydroxyapatite ceramic (HA) were implanted in femoral muscles and femoral cortical bone of dogs for 7, 14, 21, 30, 45, 60, 90, 180, and 360 days, respectively. Two dogs were used in each time point. In each dog, four cylinders (phi5x6 mm) per material were implanted in femoral muscles and 2 cylinders (phi5x6 mm) per material in femoral cortical bone. The harvested samples were processed for both histological and histomorphometric analyses. Bone was observed in BCP implanted in femoral muscles since day 30, while in HA since day 45. Quantitatively, more bone was formed in BCP than in HA at each time point after day 30 (p<0.05). The earlier and more bone formed in BCP than in HA suggests BCP a higher osteoinductive potential than HA in muscle. In femoral cortical bone defects, a bridge of bone in the defect with BCP was observed at day 21, while with HA at day 30. At days 14, 21, and 30, significantly more bone was formed in BCP than in HA (p<0.05). The results herein show that osteogenic capacities of osteoinductive materials in nonosseous tissues and osseous sites are correlated: the higher the osteoinductive potential of the material, the faster the bone repair.

Modeling BCR-ABL and MLL-AF9 leukemia in a human bone marrow-like scaffold-based xenograft model.

Although NOD-SCID IL2Rγ-/- (NSG) xenograft mice are currently the most frequently used model to study human leukemia in vivo, the absence of a human niche severely hampers faithful recapitulation of the disease. We used NSG mice in which ceramic scaffolds seeded with human mesenchymal stromal cells were implanted to generate a human bone marrow (huBM-sc)-like niche. We observed that, in contrast to the murine bone marrow (mBM) niche, the expression of BCR-ABL or MLL-AF9 was sufficient to induce both primary acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Stemness was preserved within the human niches as demonstrated by serial transplantation assays. Efficient engraftment of AML MLL-AF9 and blast-crisis chronic myeloid leukemia patient cells was also observed, whereby the immature blast-like phenotype was maintained in the huBM-sc niche but to a much lesser extent in mBM niches. We compared transcriptomes of leukemias derived from mBM niches versus leukemias from huBM-like scaffold-based niches, which revealed striking differences in the expression of genes associated with hypoxia, mitochondria and metabolism. Finally, we utilized the huBM-sc MLL-AF9 B-ALL model to evaluate the efficacy of the I-BET151 inhibitor in vivo. In conclusion, we have established human niche models in which the myeloid and lymphoid features of BCR-ABL+ and MLL-AF9+ leukemias can be studied in detail.

Parallel high-resolution confocal Raman SEM analysis of inorganic and organic bone matrix constituents.

In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.

Influence of fluoride in poly(d,l-lactide)/apatite composites on bone formation.

The influence of fluoride in poly(d,l-lactide)/apatite composites on ectopic bone formation was evaluated in sheep. Nano-apatite powders with different replacement levels of OH groups by fluoride (F) (0% (F0), 50% (F50), 100% (F100), and excessive (F200)) were co-extruded with poly (d,l-lactide) at a weight ratio of 1:1. Fluoride release from the composites (CF0, CF50, CF100, and CF200) was evaluated in vitro and bone formation was assessed after intramuscular implantation in sheep. After 24 weeks in simulated physiological solution, CF0 and CF50 showed negligible fluoride release, whereas it was considerable from the CF100 and CF200 composites. Histology showed that the incidence of de novo bone formation decreased in implants with increasing fluoride content indicating a negative influence of fluoride on ectopic bone formation. Furthermore, a significant decrease in resorption of the high fluoride-content composites and a reduction in the number of multinucleated giant cells were seen. These results show that instead of promoting, the presence of fluoride in poly(d,l-lactide)/apatite composites seemed to suppresses their resorption and osteoinductive potential in non-osseous sites.

Studying the effect of different macrostructures on in vitro cell behaviour and in vivo bone formation using a tissue engineering approach.

In the present study, we tested the in vitro process of differentiation and mineralization as well as the process of in vivo bone formation on substrates with different macrostructures. We used carbonated apatite-coated titanium discs that were respectively smooth, plasma spayed with titanium or had a porous structure. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the tissue-coated substrates were subcutaneously implanted in nude mice for 4 weeks. After 1 week of culture in the presence of the osteogenic differentiation promoter dexamethasone, the cells had formed a continuous layer of mineralized tissue on the smooth and titanium plasma-sprayed discs. In the case of the porous titanium discs, the bone-like tissue coverage was restricted to the outer surface and the peripheral pores. The influence of the macrostructure on the process of differentiation of the cultured cells depended on the presence of dexamethasone. When dexamethasone was present, the highest ALP/DNA ratios were obtained with the smooth surfaces. In the absence of dexamethasone, the highest ALP/DNA values were obtained with the rough macrostructured discs. We postulate that these different patterns were due to the shielding of cells in pits or pores of rough structured substrates by dense overlying cell layers. These cell layers are suggested to increase the exposure of excreted osteoinductive proteins and decrease the exposure of dexamethasone to underlying cells. Four weeks post-implantation, abundant bone formation could be observed on all in vitro tissue-coated substrates. The percentage of direct bone contact on the porous discs (42.3 +/- 22.3) was significantly lower compared to the non-porous discs. This was related to the process of bone infiltration into the central oriented pores that predominantly occurred in a centrifugal manner. The percentage of direct bone contact on the smooth discs (96.3 +/- 2.3) was significantly higher compared to the titanium plasma-sprayed discs (81.5 +/- 10.7). This was not due to fibrous tissue infiltration, but due to the extensive formation of bone marrow. Nevertheless, for practical reasons regarding protection of the layer of cultured cells during the implantation procedure, the use of rough or porous surface structures is suspected to be advantageous in revision surgery.

Structural arrangements at the interface between plasma sprayed calcium phosphates and bone.

Plasma sprayed coatings of tetracalcium phosphate, magnesium whitlockite and three types of hydroxyapatite, varying in degree of crystallinity, were evaluated with light microscopy, scanning electron microscopy and backscatter electron microscopy (BSE) after implantation periods of 1, 2 and 4 wk in rat femora. BSE revealed that both tetracalcium phosphate and semi-crystalline hydroxyapatite underwent distinct bulk degradation and loss of relatively large particles. Amorphous hydroxyapatite showed a gradual surface degradation, indicated by a transition zone varying in grey level between that of the coating and bone tissue, while degradation was negligible with the highly crystalline material and magnesium whitlockite. Degradation appeared to be related to bone apposition, since more bone seemed to be present on amorphous hydroxyapatite and tetracalcium phosphate, as compared to highly crystalline hydroxyapatite and magnesium whitlockite coatings. At the interface between bone and magnesium whitlockite, a seam of unmineralized bone-like tissue was frequently seen with light microscopy, while few areas with bone contact were present. X-ray microanalysis revealed that both the magnesium whitlockite coating and the unmineralized bone-like tissue contained substantial amounts of aluminium which, in addition to possible influences of magnesium, may have caused the impaired mineralization. The results of this preliminary study indicate that, with regard to early bone formation, amorphous hydroxyapatite coatings seem to be beneficial over highly crystalline coatings. However, further experiments should be performed to give conclusive data on (i) the statistical significance of the differences in bone apposition rate, and (ii) the long-term behaviour of both amorphous and highly crystalline coatings in bone and their relation to implant performance.

Poly(ether ester amide)s for tissue engineering.

Poly(ether ester amide) (PEEA) copolymers based on poly(ethylene glycol) (PEG), 1,4-butanediol and dimethyl-7,12-diaza-6,13-dione-1,18-octadecanedioate were evaluated as scaffold materials for tissue engineering. A PEEA copolymer based on PEG with a molecular weight of 300 g/mol and 25wt% of soft segments (300 PEEA 25/75) and the parent PEA polymer (0/100) sustain the adhesion and growth of endothelial cells. The in vivo degradation of melt-pressed PEEA and PEA discs subcutaneously implanted in the back of male Wistar rats was followed up to 14 weeks. Depending on the copolymer composition, a decrease in intrinsic viscosity of about 20-30% and mass loss up to 12% were measured. During the degradation process, erosion of the surface was observed by scanning electron microscopy and light microscopy. The thermal properties of the polymers during degradation were measured by differential scanning calorimetry. During the first 2 weeks, a broadening of the melting endotherm was observed, as well as an increase in the heat of fusion. Porous matrices of PEEAs and PEA could be prepared by molding mixtures of polymer and salt particles followed by leaching of the salt.

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers: effect of several additives and processing conditions.

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterials.

A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics.

With respect to the effect of material factors on calcium phosphate biomaterial-induced osteogenesis, the osteoinductive property of two kinds of porous hydroxyapatite ceramics, which were made by different producers, was investigated in dorsal muscles of dogs. One hydroxyapatite ceramic (S-HA), macroporous implants with rough pore walls containing abundant micropores, was made by Sichuan Union University (Chengdu, China); the other hydroxyapatite ceramic (J-HA), porous implants with smooth macropore walls composed of regularly aligned crystal grains, was provided by Mitsubishi Ceramic Int. (Japan). Different tissue response was detected histologically and microradiographically after the ceramic samples had been implanted in dorsal muscles of dogs for 3 and 6 months. Bone formation was found in S-HA at 3 months, which increased at 6 months. In contrast, no bone formation was detected in J-HA at both 3 and 6 months. These results indicate that with the special architecture, calcium phosphate ceramic can induce bone formation in soft tissue. As both materials were very similar in their chemical and crystallographic structures, but varied in their microstructures, the latter seem to be an important factor affecting the osteoinductive capacity of calcium phosphate ceramics. These data suggest that, by controlling the preparation of calcium phosphate ceramic, bone substitutes with intrinsic osteoinductive property can be developed from calcium phosphates.

Tissue responses of calcium phosphate cement: a study in dogs.

The in vivo properties of a new kind of calcium phosphate cement were investigated in this study. Calcium phosphate cement was implanted as paste into femoral bone and dorsal muscle of dogs for 3 and 6 months, and as prehardened form into thigh muscles of dogs for 1, 2 and 6 months. Histology was performed on thin un-decalcified sections. No foreign body reaction, no inflammation and no necrosis were found both in bony site and in muscles. There was no connective tissue layer between the cement and bone when cement paste was implanted in the bone. A creeping substitution of cement by bone, in which osteoclast-like cells resorbed the cement as if the cement is a part of bone and new bone was formed directly on the resorption line of calcium phosphate cement, was found. Bone formation was found histomorphologically in pores and deep rugged surface of cement samples (both paste and prehardened form) implanted in muscles of dogs. The induced bone was also identified with backscattered scanning electron microscopy (BSE) and by energy-dispersive X-ray micro-analysis (EDX). The results suggest that the calcium phosphate cement used in this study is biocompatible, resorbable in a manner of creeping substitution, osteoconductive and osteoinductive. It seems that an ideal bone substitute can be developed by using this type of calcium phosphate cement.

Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds.

In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations on design and assessment of outcome parameters.