The rate of vascularization of coralline hydroxyapatite

Coralline hydroxyapatite (CHAP) is a porous, biocompatible bone-graft substitute manufactured by the Replamineform process. The use of this material in the experimental and clinical settings for maxillofacial onlay grafting has been recently described. This study was designed to quantitate the rate of vascularization of coralline hydroxyapatite when used in an onlay application to membranous bone in an animal model. Sixteen onlay grafts of coralline hydroxyapatite (0.5 X 0.5 X 1.0 cm Interpore 200) were placed in a subperiosteal location on the nasal dorsum of 2- to 3-kg male New Zealand white rabbits. The grafts and nasal bones were harvested en bloc at 1, 2, 3 and 4 weeks after onlay. Prior to harvest, injectable silicone visualizing agent (Microfil*) was injected by means of carotid artery cutdown. The decalcified specimens were examined on a digitizing pad to count the number of vessels appearing in the blocks of hydroxyapatite. Counting was summed and integrated by an Apple IIe microcomputer. A significant difference (p less than 0.05) was noted in both the number of vessels and the fraction of implants infiltrated by vessels between 1 and 4 weeks. The usefulness of these previously undescribed data may be in their extrapolation to onlay grafts of coralline hydroxyapatite in maxillofacial reconstruction in humans.     

The biologic behavior of hydroxyapatite implanted into the maxillofacial skeleton

Eleven patients who previously had undergone elective facial osteotomy and in whom blocks of porous hydroxyapatite were implanted into osteotomy gaps later consented to open biopsy of the implant material. A total of 24 biopsies were harvested at a mean time of 10.2 months following implantation. Gross anatomic findings were recorded at the time of biopsy. Specimens were harvested from the zygomatic buttress of the maxilla (12), the piriform buttress of the maxilla (4), the maxillary interdental premolar region (2), and the anterior mandible (6). Histologic sections were examined undecalcified using a modified trichrome stain. Eight of the 11 patients were followed radiographically for a minimum of 24 months. In the absence of infection, there was rapid fibrovascular ingrowth and, provided there was contact with host bone, bone ingrowth. This was observed in 21 of 24 biopsy specimens. The extent of bone ingrowth, as judged qualitatively, was highly variable and did not correlate with the time interval from implantation, anatomic site, or surface area of bone-implant interface. A healing process involving an osteoid phase of bone maturation and suggestive of continued net bone production was consistently found. The gross anatomic, radiographic, and histologic findings are discussed and provide further insight into the biologic behavior of porous block hydroxyapatite implanted into the maxillofacial skeleton.     

Porous hydroxyapatite as an onlay bone-graft substitute for maxillofacial surgery

This paper chronicles 3 years of a continuing study comparing porous hydroxyapatite to autogenous bone grafts as onlays in maxillofacial surgery. Twenty-five patients, seen from June of 1984 to May of 1985, underwent onlay augmentation on various maxillary and mandibular locations. A total of 68 onlay augmentation sites comparing Interpore porous hydroxyapatite and autogenous bone were followed for 2 years or more. This long-term study compares these substances in radiologic longevity, histologic incorporation, clinical function, and aesthetic appearance.     

Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study

To assess the potential of a porous hydroxyapatite matrix to serve as a bone graft substitute, bilateral 15 X 20 mm craniectomy defects were reconstructed in 17 dogs with blocks of implant and split-rib autografts. Specimens were retrieved at 3, 6, 12, 24, and 48 months, and undecalcified sections were prepared for microscopy and histometry. The implant and graft cross-sectional areas did not change with time, documenting their equivalent ability to maintain cranial contour. Bone ingrowth extended across the implant from one cranial shelf to the other in 15 specimens. Little apparent bone ingrowth was seen in most graft specimens. Two implants and three grafts were nonunited, possibly due to lack of fixation or the orientation of the histology sections. The implant specimens were composed of 39.3 percent hydroxyapatite matrix, 17.2 percent bone ingrowth, and 43.5 percent soft-tissue ingrowth. The graft specimens were composed of 43.7 percent bone and 56.3 percent soft tissue. This study supported the thesis that a porous hydroxyapatite matrix may function in part as a bone graft substitute. The brittle hydroxyapatite matrix undoubtedly became stronger with bone ingrowth, but the degree of cranial protection achieved was not measured in this study. The size of the cranial defect used in this study did not permit estimation of the distance over which bone ingrowth may be reliably expected. There remains a need for greater understanding of the causes of nonunion, the extent of predictable ingrowth depth, and the strength of the resultant implant-bone composite.     

Porous, block hydroxyapatite as an interpositional bone graft substitute in orthognathic surgery

Forty-six nonconsecutive patients undergoing orthognathic surgery in whom blocks of coralline, porous hydroxyapatite (Interpore-200) were used in lieu of interpositional bone grafts are the subjects of this report. Surgical procedures included inferior repositioning of the maxilla (7) and chin (12), maxillary advancements in cleft (4) and noncleft individuals (13), and transverse maxillary expansions (12). Patients included in this study were only those in whom bone grafts would have been harvested had hydroxyapatite not been available. A total of 93 anatomic sites were implanted. The complication rate attributable to the use of the implant was 4.3 percent. Follow-up period ranged from 6 to 20 months, with a mean of 9.3 months. At this time, osseous stability was confirmed cephalometrically in all but two patients undergoing maxillary expansions. The biologic behavior and mechanical properties of coralline-derived, porous, block hydroxyapatite are discussed. These implant characteristics make it a feasible bone graft substitute for interpositional use in orthognathic surgery. Proper indications for its use as well as technical details to minimize complications are stressed.     

Induction of bone formation by recombinant human osteogenic protein-1 and sintered porous hydroxyapatite in adult primates

A critical issue in tissue engineering and morphogenesis of bone is the development of novel biomimetic biomaterials that are capable of optimizing the biological activity of recombinant human bone morphogenetic and osteogenic proteins, which are molecules that initiate bone formation in vivo. From a therapeutic perspective, a carrier matrix is required for the local delivery of these proteins to evoke a desired osteogenic effect. In view of the affinity of these proteins for hydroxyapatite, which may reflect the in vivo supramolecular assembly of bone proteins bound to both the extracellular matrix and the mineral component of bone, we investigated the efficacy of single applications of different doses of human osteogenic protein-1 (hOP-1) adsorbed onto sintered porous hydroxyapatites for bone induction in orthotopic calvarial defects in 12 adult male baboons (Papio ursinus) and heterotopically in the rectus abdominis of four additional baboons. In orthotopic specimens, pretreatment of sintered porous hydroxyapatites with 100 microgram of hOP-1 in 500 microliter of 5 mM hydrochloric acid resulted in rapid and diffuse osteoinduction restricted within the porous spaces of the hydroxyapatite, as evaluated by histology and histomorphometry on day 30. Hydroxyapatites treated with 500 microgram of hOP-1 showed a different pattern of bone formation and distribution on day 30 as compared with the lower dose of the recombinant morphogen. Although bone formation was extensive with the higher dose, it was found on the endocranial and pericranial aspects of the specimens, enveloping the implanted hydroxyapatite carrier, and the internal porous spaces were occupied by a rich vascular network without any bone formation. By 90 and 365 days after the implantation of both doses of hOP-1, however, there was remodelling and complete penetration of the newly induced bone within the available porous spaces. The combination of hOP-1 and hydroxyapatite also showed extensive bone formation in heterotopic specimens harvested from the rectus abdominis muscle of the baboon using doses of 5, 25, and 45 microgram of hOP-1 per implant. These findings in the adult primate demonstrate extensive bone formation by hOP-1 adsorbed onto sintered porous hydroxyapatites and suggest that predictable osteogenesis in clinical contexts for treatment of craniofacial bone defects may be engineered using inorganic, nonimmunogenic, and carvable delivery systems that initiate osteogenesis with relatively low doses of recombinant osteogenic proteins, thus mimicking the macrostructure and microstructure of living bone.     

Use of high-resolution MRI for investigation of fluid flow and global permeability in a material with interconnected porosity

We present a multi-scale experimental approach designed to improve the investigation of both localized and global fluid flow in biomaterials with randomly interconnected porosity. Coralline hydroxyapatite (ProOsteon 500 from Interpore-Cross), having a relatively well-defined porosity, was used as an in vitro model of typical bone architecture. Axial fluid velocity profiles within the pores of a cylindrical hydroxyapatite sample were characterized using high-resolution MRI in conjunction with the measurement of global flow and associated permeability based on the Darcy-type relationship. Assuming Newtonian fluid behaviour, image analysis permitted computation of local porosity, intra-pore fluid shear, and visualization of flow heterogeneity within the sample. These results may benefit applications in biomaterials for the evaluation of factors influencing bony incorporation in porous scaffolds and on porous implant and bone surfaces. Normal and diseased biological tissues are also clinical relevant applications.     

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

Nanophased porous hydroxyapatite beads with particle diameters of 25 microm and 30 microm intended for use in protein and biomolecule separation are characterized with respect to chromatographic characteristics. These particles were produced from a hydroxyapatite gel by a controlled spray process yielding microspheres containing hydroxyapatite nanocrystals. By calcification of the microspheres, nanophased porous hydroxyapatite beads were obtained. As a reference material, ceramic hydroxyapatite Types I and II with a particle diameter of 40 microm was chosen. SEM pictures show that the surface of the nanophased hydroxyapatite is very rough compared to ceramic hydroxyapatite Types I and Type II. The calcium-to-phosphorous ratio of this nanophased hydroxyapatite is 1.6, which is slightly below the theoretical ratio of 1.67 of pure hydroxyapatite. The porosity is greater than 60%. An IgG binding capacity of 60.7 mg/ml for Bio-Rad Type I and 36.0 mg/ml for Type II, 42.0 mg/ml for the nanophased material with 25 microm and 19.7 mg/ml for the nanophased material with 30 microm were observed. The nanophased material with 30 microm had the lowest mass transfer resistancy as indicated by the dependency of the dynamic binding capacity on velocity. It is assumed that the mass transport properties are characterized by a low particle diffusion resistancy or by slight intraparticle convection. The material also showed high selectivity for IgG. When culture supernatant with 5% FCS containing 3 mg/ml was loaded, pure IgG could be eluted by linear gradient with increasing sodium phosphate concentration. This nanophased material comprises a novel stationary phase for IgG separation.     

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.     

The use of coralline hydroxyapatite in a "biocomposite" free flap.

An animal model was developed to determine the feasibility of vascularizing a porous biomaterial and transferring it as part of a free flap to a recipient site with enhanced resistance to infection due to the maintenance of a blood supply. To that end, the experiment was divided into three stages. Stage 1 compared the soft-tissue response of Interpore-200 to Interpore-500, both continuously porous hydroxyapatite materials implanted beneath the panniculus carnosus of the male Sprague-Dawley rat. Pore size was an important factor as it influenced vascular ingrowth, with Interpore-200 vascularizing earlier (complete at 1 week) and more intensely. Interpore-200 was therefore used for the remainder of the experiment. After 1 week of tissue ingrowth, the implants were moved from the abdomen to the skull on a vascular pedicle as a "biocomposite" free flap. Stage 2 was the histologic evaluation of 15 "biocomposite" free flaps over various time intervals up to 8 months. The free flaps formed a fibrous union to the skull, while a simple nonvascularized Interpore-200 onlay graft (stage 2 control) demonstrated a bony union in three of four implants placed up to 2 months. Stage 3 confirmed the free flap's resistance to bacterial infection. A highly significant difference (p less than 0.005) in infection rates was demonstrated between the "biocomposite" and nonvascularized stage 3 controls with no Pseudomonas growth from 9 of 10 cultures of the free flaps 5 days after exposure to 10(4) Pseudomonas aeruginosa, while stage 3 controls demonstrated Pseudomonas growth in all cultures (heavy growth in 8 of 10). The "biocomposite" free flap has excellent potential to provide form and structure to wounds requiring reconstruction where bacterial contamination is a significant risk factor.     

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.     

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.     

Low temperature scanning electron microscopy of the superficial envelope of canine chondrocytes in culture

Low temperature scanning electron microscopy (LTSEM) has been employed to examine the surface morphology of chondrocyte cultures on ceramic granules. Well-established cultures on porous hydroxyapatite consist of ceramic cores overlaid and interspersed with a cellular matrix of collagen and proteoglycan (Cheung, 1985); of especial interest is the superficial layer of cells. These cells are believed, on the basis of immuno-light microscopy (Gardner et al., 1987), to be coated by an hydrated porous envelope of collagen/proteoglycan which is likely to obscure cell outlines. This relationship is confirmed by enzymic digestion of the superficial material. Post-digestion LTSEM examination of the fully hydrated preparations establishes the existence of arrays of rounded structures identified as superficial cells.     

Assessment of the effects on growth of porous hydroxyapatite granule cranioplasty in the immature guinea pig craniofacial skeleton

The immature guinea pig was used to study the effects on growth of porous granular hydroxyapatite used as an onlay cranioplasty and inlay cranioplasty to reconstruct full-thickness cranial defects in a growing craniofacial skeleton. Forty Hartley guinea pigs, 20 immature animals and 20 mature animals, were divided into four groups each containing five mature and five immature animals. The mature animals served as controls. Group I underwent elevation and replacement of the parietal periosteum. Group II underwent placement of hydroxyapatite between periosteum and parietal bone. Group III underwent elevation and replacement of autogenous bone flap after the formation of a 1 x 1-cm craniectomy defect in the parietal skull. Group IV underwent elevation of a 1 x 1-cm parietal craniectomy and reconstruction of the defect with hydroxyapatite granules placed between the dura and periosteum. Immature animals were killed at maturity at 3.5 months and mature animals were killed 2.5 months postoperatively. Macroscopic examination of the operative field, transverse and longitudinal cephalometric measurements, and histological sections encompassing the operative sites were compared. Macroscopically, all reconstructed operative sites were fully incorporated into the cranium. Histological staining of the sectioned operative site revealed no hydroxyapatite migration through the cranial bone or dura. No inflammatory or foreign body reaction was evident in the subcutaneous tissue, periosteum, or dura. No statistically significant cephalometric intergroup or intragroup differences were found at the conclusion of the study. The results of this study indicate that a granular porous form of hydroxyapatite may be used as an onlay or inlay cranioplasty in the immature guinea pig craniofacial skeleton without evidence of dural inflammation, granule migration, or growth restriction or retardation.     

Mechanical response of porous scaffolds for cartilage engineering

Mechanical properties of scaffolds seeded with mesenchymal stem cells used for cartilage repair seem to be one of the critical factors in possible joint resurfacing. In this paper, the effect of adding hyaluronic acid, hydroxyapatite nanoparticles or chitosan nanofibers into the cross-linked collagen I on the mechanical response of the lyophilized porous scaffold has been investigated in the dry state at 37 oC under tensile loading. Statistical significance of the results was evaluated using ANOVA analysis. The results showed that the addition of hyaluronic acid significantly (p<0.05) reduced the tensile elastic modulus and enhanced the strength and deformation to failure of the modified cross-linked collagen I under the used test conditions. On the other hand, addition of hydroxyapatite nanoparticles and chitosan nanofibers, respectively, increased the elastic modulus of the modified collagen ten-fold and four-fold, respectively. Hydroxyapatite caused significant reduction in the ultimate deformation at break while chitosan nanofibers enhanced the ultimate deformation under tensile loading substantially (p<0.05). The ultimate tensile deformation was significantly (p<0.05) increased by addition of the chitosan nanofibers. The enhanced elastic modulus of the scaffold was translated into enhanced resistance of the porous scaffolds against mechanical load compared to scaffolds based on cross-linked neat collagen or collagen with hyaluronic acid with similar porosity. It can be concluded that enhancing the rigidity of the compact scaffold material by adding rigid chitosan nanofibers can improve the resistance of the porous scaffolds against compressive loading, which can provide more structural protection to the seeded mesenchymal stem cells when the construct is implanted into a lesion. Moreover, scaffolds with chitosan nanofibers seemed to enhance cell growth compared to the neat collagen I when tested in vitro as well as the scaffold stability, extending its resorption to more than 10 weeks.     

Impact of the porous microstructure on the overall elastic properties of the osteonal cortical bone

Mechanical properties of bones are largely determined by their microstructure. The latter comprises a large number of diverse pores. The present paper analyzes a connection between structure of the porous space of the osteonal cortical bone and bone's overall anisotropic elastic moduli. The analysis is based on recent developments in the theory of porous materials that predict the anisotropic effective moduli of porous solids in terms of pores' shapes, orientations and densities. Bone's microstructure is modeled using available micrographs. The calculated anisotropic elastic constants for porous cortical bone are, mostly, in agreement with available experimental data. The influence of each of the pore types on the overall moduli is examined. The results of the analysis can also be used to estimate the extent of mineralization (hydroxyapatite content) if the overall porosity and the effective moduli are known and, vice versa, to estimate porosity from the measured moduli and the extent of mineralization.     

Tensile strength of bone fixation of hydroxyapatite coated Schanz screws of the Heidelberg External Fixation System (HEFS)--comparative torque measurements in clinical use and in cadaver tibia]

It is claimed in the literature that hydroxyapatite(HA)-coated screws of external fixators have superior fixation strength in bone, which is postulated to lead to a substantial decrease in loosening and infection rates. We report on a study of the maximum torque values developed while inserting and removing 30 HA-coated Schanz screws of 8 Heidelberg external fixation systems applied to the tibia to correct leg length differences and axial deformities. The infection rate was determined in accordance with defined criteria, and was found to be about 20% for the HA-coated screws. Screws without infection showed an extraction torque above insertion torque, screws with infection an extraction torque below. A significant correlation (p = 0.05) was seen between infection and decrease in fixation strength (quotient: loosening torque/tightening torque). To exclude the impact of such biological processes as osteointegration and bone remodelling, the clinical results were compared with the torques measured for coated and uncoated Schanz screws in a human cadaveric tibia. A significantly higher fixation strength in bone was found for HA-coated screws in comparison with uncoated screws (p = 0.002). These data warrant a clinical study directly comparing HA-coated and uncoated Schanz screws.     

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.     

Cuttlefish bone scaffold for tissue engineering: a novel hydrothermal transformation, chemical-physical, and biological characterization

Purpose: Natural resources are receiving growing interest because of their possible conversion from a cheap and easily available material into a biomedical product. Cuttlefish bone from Sepia Officinalis was investigated in order to obtain an hydroxyapatite porous scaffold using hydrothermal transformation. Methods: Complete conversion of the previous calcium carbonate (aragonite) phase into a calcium phosphate (hydroxyapatite) phase was performed with an hydrothermal transformation at 200 °C (~ 15 atm), for four hours, with an aqueous solution of KH2PO4 in order to set the molar ratio Ca/P = 10/6 in a reactor (Parr 4382). The complete conversion was then analyzed by TGA, ATR-FTIR, x-ray diffraction, and SEM. Moreover, the material was biologically investigated with MC3T3-E1 in static cultures, using both osteogenic and maintenance media. The expression of osteogenic markers as ALP and osteocalcin and the cell proliferation were investigated. Results: Cuttlefish bone has been successfully transformed from calcium carbonate into calcium phosphate. Biological characterization revealed that osteogenic markers are expressed using both osteogenic and maintenance conditions. Cell proliferation is influenced by the static culture condition used for this three-dimensional scaffold. Conclusions: The new scaffold composed by hydroxyapatite and derived for a natural source presents good biocompatibility and can be used for further investigations using dynamic cultures in order to improve cell proliferation and differentiation for bone tissue engineering.