Bone morphogenetic proteins in craniofacial and periodontal tissue engineering: experimental studies in the non-human primate Papio ursinus

The bone morphogenetic and osteogenic proteins (BMPs/OPs), pleiotropic members of the transforming growth factor-beta (TGF-beta) supergene family act as soluble signals for the de novo initiation of bone formation, sculpting the multicellular mineralized structures of the bone-bone marrow organ. The strikingly pleiotropic effects of BMPs/OPs spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. BMPs/OPs are the common molecular initiators deployed for embryonic development and the induction of bone formation and regeneration in postnatal osteogenesis. Naturally derived BMPs/OPs extracted and purified from baboon and bovine bone matrices induce complete regeneration of non-healing calvarial defects in the non-human primate Papio ursinus as well as the induction of cementogenesis and the morphogenesis of a periodontal ligament system with a faithful insertion of Sharpey's fibers into the newly formed cementum. gamma-Irradiated recombinant human osteogenic protein-1 (hOP-1) delivered by xenogeneic bovine collagenous bone matrices completely regenerated and maintained the architecture of the induced bone after treatment of calvarial defects with single applications of doses of 0.1, 0.5 and 2.5mg hOP-1 per gram of carrier matrix. The long-term implantation of hOP-1 delivered by gamma-irradiated bovine bone matrices induced the regeneration of the three essential components of the periodontium, i.e. cementum, periodontal ligament and alveolar bone. The osteogenic proteins of the TGF-beta superfamily are sculpting tissue constructs that engineer skeletal tissue regeneration in molecular terms. The pleiotropy of the signalling molecules of the TGF-beta superfamily is highlighted by the redundancy of molecular signals initiating bone formation, including the TGF-beta isoforms per se, powerful inducers of endochondral bone formation but in the primate only. The induction of bone develops a mosaic structure in which members of the TGF-beta superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.     

Osteogenin, a bone morphogenetic protein, adsorbed on porous hydroxyapatite substrata, induces rapid bone differentiation in calvarial defects of adult primates.

Osteogenin, a bone morphogenetic protein, in conjunction with insoluble collagenous bone matrix initiates local endochondral bone differentiation by induction in vivo. This study, by exploiting the affinity of native osteogenin for hydroxyapatite, was designed to construct a delivery system for the expression of the biologic activity of osteogenin in nonhealing calvarial defects of adult primates. After exposure of the calvaria, 64 cranial defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). Defects were implanted with disks of porous nonresorbable and resorbable hydroxyapatite substrata obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. In each animal, one disk of each hydroxyapatite preparation was treated with osteogenin isolated and purified from baboon bone matrix after sequential chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200 gel filtration columns. The remaining two defects were implanted with one disk of each hydroxyapatite preparation without osteogenin as control. Histomorphometry on decalcified sections prepared on days 30 and 90 showed superior osteogenesis in osteogenin-treated nonresorbable hydroxyapatite specimens as compared with controls. On day 90, substantial bone formation also had occurred in control nonresorbable hydroxyapatite specimens. On day 90, but not on day 30, significantly greater amounts of bone had formed in osteogenin-treated resorbable specimens as compared with resorbable controls. Overall, resorbable substrata performed poorly when compared with nonresorbable substrata, perhaps due to a premature dissolution of the implants. These results provide evidence that the biologic activity of osteogenin can be restored and delivered by a substratum other than the organic collagenous matrix, inducing rapid bone differentiation in calvarial defects of adult nonhuman primates. The adsorption strategy of osteogenin on porous inorganic nonimmunogenic substrata may help to design appropriate osteogenic delivery systems for craniofacial and orthopedic applications in humans.     

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.     

Induction of bone in composites of osteogenin and porous hydroxyapatite in baboons.

A major goal of the combined effort of basic scientists and plastic and reconstructive surgeons is the development of novel bone substitutes based on osteogenic growth and differentiation factors with optimal delivery systems for skeletal repair. Osteogenin is a protein initiator of bone differentiation. The present study examined the osteogenic potential of osteogenin in combination with porous hydroxyapatite replicas obtained after hydrothermal conversion of calcium carbonate exoskeletons of corals. Bovine osteogenin, with an apparent molecular weight of 28 to 42 kDa, purified by hydroxyapatite-Ultrogel adsorption chromatography, heparin-Sepharose affinity chromatography, and HR Sephacryl S-200 molecular sieve chromatography, was delivered into rods of nonresorbable and resorbable hydroxyapatite replicas with an average porosity of 600 microns. A total of 48 rods were bioassayed for osteogenic activity by intramuscular implantation into eight adult baboons (Papio ursinus) as a prerequisite for clinical trials in humans. Bovine osteogenin fractions reconstituted with baboon insoluble collagenous bone matrix were implanted in an additional four adult baboons. Specimens were harvested at 30 and 90 days after implantation and subjected to histomorphometry and alkaline phosphatase activity determination. Differentiation of bone occurred in nonresorbable hydroxyapatite rods, both osteogenin-treated and controls. However, no bone formation was observed in resorbable rods, even in the presence of osteogenin. These results demonstrate that the surface and chemical characteristics of the substratum, independent of the osteogenic stimulus, have a profound influence on the morphogenesis of bone. The demonstration of bone induction in nonhuman primates with porous nonresorbable hydroxyapatite replicas and baboon insoluble collagenous bone matrix reconstituted with bovine osteogenin establishes the therapeutic potential of the principle of bone induction in craniofacial, periodontal, and orthopedic reconstructive surgery.     

Soluble, insoluble and geometric signals sculpt the architecture of mineralized tissues

Bone morphogenetic and osteogenic proteins (BMPs/OPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are soluble mediators of tissue morphogenesis and induce de novo endochondral bone formation in heterotopic extraskeletal sites as a recapitulation of embryonic development. In the primate Papio ursinus, the induction of bone formation has been extended to the TGF-beta isoforms per se. In the primate and in the primate only, the TGF-beta isoforms are initiators of endochondral bone formation by induction and act in a species-, site- and tissue-specific mode with robust endochondral bone induction in heterotopic sites but with limited new bone formation in orthotopic bone defects. The limited inductive capacity orthotopically of TGF-beta isoforms is associated with expression of the inhibitory Smads, Smad6 and Smad7. In primates, bone formation can also be induced using biomimetic crystalline hydroxyapatite matrices with a specific surface geometry and without the exogenous application of osteogenic proteins of the TGF-beta superfamily, even when the biomimetic matrices are implanted heterotopically in the rectus abdominis muscle. The sequence of events that directs new bone formation upon the implantation of highly crystalline biomimetic matrices initiates with vascular invasion, mesenchymal cell migration, attachment and differentiation of osteoblast-like cells attached to the substratum, expression and synthesis of osteogenic proteins of the TGF-beta superfamily resulting in the induction of bone as a secondary response. The above findings in the primate indicate enormous potential for the bioengineering industry. Of particular interest is that biomimetic matrices with intrinsic osteoinductivity would be an affordable option in the local context.     

The induction of endochondral bone formation by transforming growth factor-beta(3): experimental studies in the non-human primate Papio ursinus.

Transforming growth factor-beta(3) (TGF-beta(3)), a multi-functional growth modulator of embryonic development, tissue repair and morphogenesis, immunoregulation, fibrosis, angiogenesis and carcinogenesis, is the third mammalian isoform of the TGF-beta subfamily of proteins. The pleiotropism of the signalling proteins of the TGF-beta superfamily, including the TGF-beta proteins per se, are highlighted by the apparent redundancy of soluble molecular signals initiating de novo endochondral bone induction in the primate only. In the heterotopic bioassay for bone induction in the subcutaneous site of rodents, the TGF-beta(3) isoform does not initiate endochondral bone formation. Strikingly and in marked contrast to the rodent bioassay, recombinant human (h)TGF-beta(3), when implanted in the rectus abdominis muscle of adult non-human primates Papio ursinus at doses of 5, 25 and 125 mug per 100 mg of insoluble collagenous matrix as carrier, induces rapid endochondral bone formation resulting in large corticalized ossicles by day 30 and 90. In the same animals, the delivery of identical or higher doses of theTGF-beta(3) protein results in minimal repair of calvarial defects on day 30 with limited bone regeneration across the pericranial aspect of the defects on day 90. Partial restoration of the bone induction cascade by the hTGF-beta(3) protein is obtained by mixing the hTGF-beta(3) device with minced fragments of autogenous rectus abdominis muscle thus adding responding stem cells for further bone induction by the hTGF-beta(3) protein. The observed limited bone induction in hTGF-beta(3)/treated and untreated calvarial defects in Papio ursinus and therefore by extension to Homo sapiens, is due to the influence of Smad-6 and Smad-7 down-stream antagonists of the TGF-beta signalling pathway. RT-PCR, Western and Northern blot analyses of tissue specimens generated by the TGF-beta(3) isoform demonstrate robust expression of Smad-6 and Smad-7 in orthotopic calvarial sites with limited expression in heterotopic rectus abdominis sites. Smad-6 and -7 overexpression in hTGF-beta(3)/treated and untreated calvarial defects may be due to the vascular endothelial tissue of the arachnoids expressing signalling proteins modulating the expression of the inhibitory Smads in pre-osteoblastic and osteoblastic calvarial cell lines controlling the induction of bone in the primate calvarium.     

Fibroblast growth factor: effects on osteogenesis and chondrogenesis in the chick embryo.

In vivo effects of fibroblast growth factor (FGF) on osteogenesis were evaluated in the chick embryo. Autoradiographic studies of 3H-proline labeling over bone matrix indicated that 24 h after treatment on day 11, FGF stimulated osteogenic cell proliferation, while inhibiting the production of bone matrix collagen. However, 4 days after multiple doses of FGF, the large pool of newly formed osteogenic and chondrogenic cells expressed their function with the increased formation of matrix. The data provide in vivo evidence of the effects of exogenous FGF on osteogenesis and also point to a possible role for FGF both in embryonic osteogenesis and in fracture repair.     

Effect of Dual Treatment with SDF-1 and BMP-2 on Ectopic and Orthotopic Bone Formation

Purposes

The potent stem cell homing factor stromal cell-derived factor-1 (SDF-1) actively recruits mesenchymal stem cells from circulation and from local bone marrow. It is well established that bone morphogenetic protein-2 (BMP-2) induces ectopic and orthotopic bone formation. However, the exact synergistic effects of BMP-2 and SDF-1 in ectopic and orthotopic bone regeneration models have not been fully investigated. The purpose of this study was to evaluate the potential effects of simultaneous SDF-1 and BMP-2 treatment on bone formation.

Materials and Methods

Various doses of SDF-1 were loaded onto collagen sponges with or without BMP-2.These sponges were implanted into subcutaneous pockets and critical-size calvarial defects in C57BL/6 mice. The specimens were harvested 4 weeks post-surgery and the degree of bone formation in specimens was evaluated by histomorphometric and radiographic density analyses. Osteogenic potential and migration capacity of mesenchymal cells and capillary tube formation of endothelial cells following dual treatment with SDF-1 and BMP-2 were evaluated with in vitro assays.

Results

SDF-1-only-treated implants did not yield significant in vivo bone formation and SDF-1 treatment did not enhance BMP-2-induced ectopic and orthotopic bone regeneration. In vitro experiments showed that concomitant use of BMP-2 and SDF-1 had no additive effect on osteoblastic differentiation, cell migration or angiogenesis compared to BMP-2 or SDF-1 treatment alone.

Conclusions

These findings imply that sequence-controlled application of SDF-1 and BMP-2 must be further investigated for the enhancement of robust osteogenesis in bone defects.     

The roles of circRFWD2 and circINO80 during NELL‐1‐induced osteogenesis

Bone defects caused heavy social and economic burdens worldwide. Nel‐like molecule, type 1 (NELL‐1) could enhance the osteogenesis and the repairment of bone defects, while the specific mechanism remains to be elucidated. Circular RNAs (circRNAs) have been found to play critical roles in the tissue development and serve as biomarkers for various diseases. However, it remains unclear that the expression patterns of circRNAs and the roles of them played in recombinant NELL‐1‐induced osteogenesis of human adipose‐derived stem cells (hASCs). In this study, we performed RNA‐sequencing to investigate the expression profiles of circRNAs in recombinant NELL‐1‐induced osteogenic differentiation and identified two key circRNAs, namely circRFWD2 and circINO80. These two circRNAs were confirmed to be up‐regulated during recombinant NELL‐1‐induced osteogenesis, and knockdown of them affected the positive effect of NELL‐1 on osteogenesis. CircRFWD2 and circINO80 could interact with hsa‐miR‐6817‐5p, which could inhibit the osteogenesis. Silencing hsa‐miR‐6817‐5p could partially reverse the negative effect of si‐circRFWD2 and si‐circINO80 on the osteogenesis. Therefore, circRFWD2 and circINO80 could regulate the expression of hsa‐miR‐6817‐5p and influence the recombinant NELL‐1‐induced osteogenic differentiation of hASCs. It opens a new window to better understanding the effects of NELL‐1 on the osteogenic differentiation of hASCs and provides potential molecular targets and novel methods for bone regeneration efficiently and safely.     

In vitro osteogenic differentiation of human ES cells

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.     

Three-dimensional cellular development is essential for ex vivo formation of human bone

Tissue engineering of human bone is a complex process, as the functional development of bone cells requires that regulatory signals be temporally and spatially ordered. The role of three-dimensional cellular interactions is well understood in embryonic osteogenesis, but in vitro correlates are lacking. Here we report that in vitro serum-free transforming growth factor (TGF)-beta1 stimulation of osteogenic cells immediately after passage results in the formation of three-dimensional cellular condensations (bone cell spheroids) within 24 to 48 hours. In turn, bone cell spheroid formation results in the up-regulation of several bone-related proteins (e.g., alkaline phosphatase, type I collagen, osteonectin) during days 3-7, and the concomitant formation of micro-crystalline bone. This system of ex vivo bone formation should provide important information on the physiological, biological and molecular basis of osteogenesis.     

Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo

Some members of the bone morphogenetic protein subfamily (BMP-2 and -7) are currently used in orthopedic surgery for several applications. Although their use is considered safe at short term, the high doses of growth factors needed make these treatments expensive and their safety uncertain at long term. BMP-6 has been much less studied than BMP-2 and -7, but some authors suggest that this BMP might have a stronger osteogenic activity than the previously mentioned. Having in mind that angiogenesis plays a well-known role during bone formation, the aim of this work was to study the effect of combining BMP-6 with bFGF on both the growth and differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs), as well as on in vivo osteogenesis. We demonstrate that a low dose of bFGF enhances the osteogenic differentiation of MSCs induced by BMP-6 in vitro. Furthermore, we also demonstrate that bone formation in vivo induced by BMP-6 can be accelerated and enhanced by adding a low dose of bFGF, what might suggest a synergic effect between these growth factors on in vivo osteogenesis.     

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.     

Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic Differentiation and Bone Regeneration In Vitro and In Vivo

Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.     

SEMA3B-AS1-inhibited osteogenic differentiation of human mesenchymal stem cells revealed by quantitative proteomics analysis

Human mesenchymal stem cells (hMSCs) are fibroblastoid multipotent adult stem cells with capacities of differentiation into osteoblasts and chondrocytes and show great potential in new bone formation and bone repair-related clinical settings, such as osteoporosis. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in various biological processes. Here, we report an antisense lncRNA SEMA3B-AS1 regulating hMSCs osteogenesis. SEMA3B-AS1 is proximal to a member of the semaphorin family Sema3b. Overexpression of SEMA3B-AS1 using the lentivirus system markedly inhibits the proliferation of hMSCs and meanwhile reduces osteogenic differentiation. Using a comprehensive proteomic technique named isobaric tag for relative and absolute quantitation, we found that SEMA3B-AS1 significantly alters the process of osteogenesis through downregulating the expression of proteins involved in actin cytoskeleton, focal adhesion, and extracellular matrix–receptor interaction, while increasing the expression of proteins in the spliceosome. Collectively, we find that SEMA3B-AS1 is a target for controlling osteogenesis of hMSCs.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.     

Surface energetics of bone mineral and synthetic hydroxyapatite using inverse gas chromatography

Surface energy is one of the important factors that govern protein adhesion and cell attachment on biomaterial surfaces. Inverse gas chromatography (IGC) provides an excellent method to measure the surface energetics of rough and porous biosurfaces. In this study IGC was used to characterize and compare the surface energetics of synthetic and biological hydroxyapatites (natural bone mineral). IGC experiments were performed on three samples: synthetic hydroxyapatites with two levels of purity (99% and 90%) and natural biological hydroxyapatite obtained from bovine trabecular bone. The Lifshitz-Van der Waals component of the surface free energy (γ(S)(LW)) and specific interaction parameter (?(π)) were determined by using homologous series of n-alkanes and alkenes as IGC probe molecules, respectively. The synthetic hydroxyapatite had values of γ(S)(LW) of 33.4 mJm?2 at 99% purity and 53.3 mJm?2 at 90% purity. Biological hydroxyapatite had a value of γ(S)(LW) of 45.7 mJm?2. For the synthetic hydroxyapatite, the values of π-bond specific interaction parameters, ?(π), were 0.95 mJ (99%) and 3.01 mJ (90%). The biological hydroxyapatite sample had a value of 2.44 mJ for ?(π). The results suggest that, as compared to the synthetic compounds, the biological apatite has considerable surface heterogeneity, either chemical (impurities) or structural suggesting a scaffold surface that is more conducive of protein adhesion and cell attachment.