Coralline bone graft substitutes.

Coralline porous ceramics are biocompatible and osteoconductive implants. They have proven to be effective as bone graft substitutes in large animal models and in humans. Bone and supporting soft tissue grow into and throughout their porosity if the implant is placed in direct apposition to viable bone and the interfaces are stabilized. The bone within the implant remodels in response to Wolff's law. Both the implant properties (chemistry and porosity) and the biologic environment modulate the rate of implant resorption. Composite technology with resorbable polymers can improve the mechanical properties of these ceramics.

Coralline hydroxyapatite bone graft substitutes. Evaluation of bone density with dual energy x-ray absorptiometry.

RATIONALE AND OBJECTIVES: The authors evaluate whether dual-energy x-ray absorptiometry (DXA) is a reliable method to determine the density of natural coralline hydroxyapatite (HA) blocks used as bone graft substitutes. METHODS: To evaluate the basic density of HA blocks from the same coral heads with and without titanium meshes, densitometry of 12 HA-500 blocks (genus Goniopora) and 12 HA-200 blocks (genus Porites) was performed. In addition, density measurements of 30 HA blocks (HA-500, n = 15; HA-200, n = 15) from different coral heads were obtained to assess if the originating coral head influences the basic density of blocks within one coral genera. To assess standard deviation serial measurements on eight coralline HA blocks, four with titanium meshes and four without were performed. In the ex vivo study, densitometry of 12 HA blocks (HA-500, n = 4; HA-200, n = 8) used as bone graft substitutes in the mandibles and craniums of adult mongrel dogs was performed. Densities were measured after bone ingrowth for 2 and 4 months, respectively. All measurements were obtained with a Lunar DPX with scan mode "slow 750" in the spine program with the regions-of-interests selected manually. Bone ingrowth was assessed by computer-assisted histomorphometry, which was considered the gold standard. Statistical analysis was performed to correlate the densities of plain HA blocks with and without meshes to the specific weights of the blocks. RESULTS: Significant positive correlation was found between the density of each HA block (both coral species) with and without meshes and the calculated specific weights. Densitometry values showed no significant differences depending on the originating coral heads. Standard deviation ranged between +/- 3.8% and +/- 4.1% (HA-500) and between +/- 3.0% and +/- 3.8% (HA-200). Hydroxyapatite-500 blocks showed marked increased densities between 15% and 34% after 4 months in three specimens in which bone ingrowth between 16.9% and 21.1% was revealed by histomorphometry; no increase of density was observed in one specimen, which presented only minimal bone ingrowth and signs of infection. Despite bone invasion between 12% and 25.8%, no increased densities were observed for HA-200 implants. CONCLUSIONS: Dual-energy x-ray absorptiometry is an accurate and reproducible modality to assess the densities of plain coralline HA blocks and to monitor bone ingrowth into coralline HA-500 but not into HA-200 block implants.

Bone formation and implant degradation of coralline porous ceramics placed in bone and ectopic sites.

PURPOSE: This study was undertaken to determine the resorption rate of porous ceramic implants. The hypothesis was that implants placed in soft tissues would degrade more rapidly than implants placed in bone. MATERIALS AND METHODS: To test this hypothesis, implants were manufactured by applying a thin coating of hydroxylapatite onto an interconnected, porous calcium carbonate substrate. Control implants were made entirely of hydroxylapatite with identical microstructure. Two adult dogs received a total of 56 implants placed in the femur, skeletal muscle, and subcutaneous tissues. After killing the animals at 4 months, the specimens were removed, embedded in plastic, sectioned, and either stained for light microscopic examination or subjected to quantitative image analysis using a scanning electron microscope. RESULTS: Contrary to the hypothesis, the rate of degradation was faster for implants placed in bone than in soft tissue. Within the 4 months, degradation was 24% to 63% in bone, depending on the composition. However, it was not statistically significant in either intramuscular or subcutaneous tissue. A surprising observation was that bone ingrowth occurred in 67% of the implants placed in soft tissues. On average, it was 4.3% in intramuscular sites and 6.6% in subcutaneous sites. This bone was histologically normal in 71% of the implants containing bone. CONCLUSION: This study demonstrates that porous ceramic implants composed of hydroxylapatite on calcium carbonate will degrade more rapidly in bone defects than in soft tissue sites. In addition, implants with interconnected porosity and surfaces of hydroxylapatite will become ingrown with bone even after placement in soft tissues. The exact mechanisms for both of these phenomena are not understood.

Effect of demineralized bone matrix on bone growth within a porous HA material: a histologic and histometric study.

Coralline hydroxyapatite (cHA) is an osteoconductive material currently being used as a bone graft substitute. Created by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, this material has a porous structure similar to cancellous bone. Addition of demineralized bone matrix (DBM) would conceivably create a composite with both osteoconductive and osteoinductive properties. This pilot study evaluated the healing of rabbit cranial defects that had been filled with cHA or cHA augmented with a DBM gel formed by adding glycerol to the DBM particulate. Data from these were then compared to unfilled defects from a previous study. Results indicated enhancement of new bone formation and an increase in the rate of healing in the defects filled with the cHA-DBM gel composite. Further studies are warranted.

An in-vitro evaluation of coralline porous hydroxyapatite as a scaffold for osteoblast growth.

The purpose of this study was to determine the potential of coralline calcium phosphate ceramics to support osteoblast growth for a proposed bone-ceramic composite for skeletal tissue repair. The goal was the development of a matrix with both osteogenic and osteoconductive properties, as compared to ceramic alone, which is solely osteoconductive. MC3T3-E1 osteoblast-like cells were seeded onto sintered and non-sintered porous coralline hydroxyapatite (HA), and onto non-porous hydroxyapatite discs. These in-vitro studies demonstrated that coralline HA supported the growth of osteoblast-like cells. Porous discs supported higher numbers of cells than non-porous discs. Sintering encouraged cell growth, with higher numbers of cells adhered to sintered porous HA discs by day seven. The results suggest that HA can provide a support for osteoblast cells as part of a matrix which may prove to be osteogenic in vivo and may, accordingly, enhance the bone repair process.

Bone ingrowth and mechanical properties of coralline hydroxyapatite 1 yr after implantation.

A previous study of coralline hydroxyapatite as a bone-graft substitute was extended from 4 to 12 months to determine better the relationships between implantation time, bone ingrowth and mechanical properties. The model consisted of a 10 x 30 mm window defect in the shaft of the canine radius (a cortical site), and a 10 mm diameter cylindrical defect in the head of the humerus (a cancellous site). In the new study, these two defects were made bilaterally in eight dogs, and filled with block-form coralline hydroxyapatite. The radius defects were supported by a metal fixation plate which was removed after 9 months. After 12 months, the dogs were killed and the left-side implants were analyzed histomorphometrically and mechanically. The right-side radius and humerus were reserved for structural analysis. The results were combined with those previously measured after 4, 8, 12 and 16 wk of implantation. In the cortical site, bone ingrowth increased from 52% at 16 wk to 74% at 1 yr. In the cancellous site, bone ingrowth was 38% after 4 wk, then fell monotonically, reaching 17% at 1 yr. Bending and compressive strength and stiffness of the radius implants increased throughout the post-implantation year, but compressive strength and stiffness of the humerus implants did not change after the first 2-4 months. Mechanical properties were strongly correlated to bone ingrowth in the cortical, but not the cancellous, site. The volume fraction of the coralline hydroxyapatite material diminished significantly with time in the cortical, but not the cancellous, site.

Bone formation process in porous calcium carbonate and hydroxyapatite.

This study determined the bone formation in porous calcium carbonate (CC) and porous hydroxyapatite (HA) in ectopic sites. The bone formation stimulus was derived from bone marrow cells. CC and HA in the shape of disks were implanted with or without rat marrow cells into subcutaneous sites of syngeneic rats. The CC and HA had identical microstructure: pore size was 190-230 microns, porosity was 50-60% and they were fully interconnected. Bone did not form in any implants without marrow cells (disks themselves), whereas bone consistently formed in the pores of all implants with marrow cells after 4 weeks. The bone formation of both CC and HA occurred initially on surface of the pore regions and progressed toward the center of the pore. Scanning electron microscopy and electron-probe microanalysis revealed a continuum of calcium at the interfaces of both bone/CC and bone/HA implants. These results indicate that the bone formation in calcium carbonate derived from marine corals is comparable to the bioactive hydroxyapatite.

Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation.

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.

Effects of bone ingrowth on the strength and non-invasive assessment of a coralline hydroxyapatite material.

The dependence of strength on the amount of bone growth into a hydroxyapatite material made from coral was investigated. Block and granular forms of the material were implanted into cortical and trabecular regions of the skeletons of 16 dogs. The results were examined after 4, 8, 12 and 16 wk, with four dogs in each experimental group. When implanted into cortical bone, the bending strength of the implant material was found to be highly correlated with the amount of pore space which had become occupied by bone (r = 0.92, P less than 0.005 for the block form; r = 0.84, P less than 0.005 for the granular form). Multiple regression analysis showed that six histomorphometric measures of ingrowth accounted for 96% of the variability in bending strength of the block material, and there were no significant differences between block and granular forms of the material. On the other hand, when implanted into trabecular bone, the block form of the material achieved greater compressive strength than the granular form. While both strength and ingrowth increased with time, there were poor correlations between these two variables. Finally, when the material is implanted into trabecular bone, it becomes stronger in compression than the surrounding bone; when implanted in cortical bone, linear modelling suggests that resorption and replacement of the implant would be required to approximate the bending strength of the surrounding bone.

Preservation of compliance in a small diameter microporous, silicone rubber vascular prosthesis.

Compliance is believed to be a significant factor in maintaining the patency of small diameter vascular grafts. This study evaluated the compliance changes with time of microporous Replamineform silicone rubber prostheses. The compliance of 15 canine femoral artery interpositions (4 mm internal diameter X 6 cm length) was measured by in-vivo electromagnetic rheoangiometry immediately following implantation and at intervals to eight months. At implantation, silicone rubber grafts were overcompliant (15.0 +/- 1.1% radial change/mmHg X 10(-2); mean +/- S.E.) compared to the proximal artery (7.7 +/- .6%). The compliance of the prostheses decreased within two weeks (6.3 +/- .9%) and remained isocompliant to the proximal artery for eight months. The compliance determinations for the silicone rubber grafts were compared with those from PTFE prostheses and vein grafts acquired by the same method in a previous study. The analysis demonstrates the preservation of isocompliance of the silicone rubber prostheses compared to the native arteries. In contrast, the minimally compliant vein grafts and PTFE prostheses continued to decrease in compliance following implantation. This microporous silicone rubber graft may improve the success of small internal diameter arterial reconstructions by eliminating failures caused by compliance mismatch between the artery and the prosthesis.

Ventilatory responses to partial cardiopulmonary bypass at rest and exercise in dogs.

We determined the role of blood flow-induced changes in CO2 load to the lungs on ventilatory control, at rest and in the steady-state of electrically induced exercise, in the anesthetized dog. A portion of the vena caval blood was diverted to the descending aorta following "arterialization" through an extracorporeal gas exchanger. Ventilation typically decreased, both at rest and during exercise (i.e., at 2 different levels of mixed venous CO2), in proportion to the CO2 loss; arterial PCO2 was consequently regulated. There were concomitant increases of the pulmonary and peripheral vascular resistance. Bilateral cervical vagosympathectomy markedly attenuated the ventilatory response at rest, thus disrupting arterial PCO2 homeostasis, but not so during exercise. The results therefore provide evidence for and support the suggestion of CO2 flow-related hyperpnea both at rest and during muscular exercise.

Biomaterial aspects of Interpore-200 porous hydroxyapatite.

Interpore-200 is the product of over 11 years of continuous research and development. It has been investigated at over 25 research centers in a wide variety of animal and human implant settings, including alveolar ridge augmentation, periodontics, and orthognathic reconstructions. The biomaterial aspects of Interpore-200 show the following: Interpore-200 has a highly interconnected, three-dimensional porosity that is uniform and consistent. The hydroxyapatite manufactured from marine corals is biocompatible and nontoxic. Interpore-200 is essentially pure hydroxyapatite, with the balance consisting of tricalcium phosphate. Interpore-200 is approximately 55 to 65 per cent porous with nominal pore diameters of 200 micron. Unlike nonporous materials, Interpore-200 is osteoconductive and results, when placed next to a viable bone, in an advancing front of bone into the implant. From 50 to 88 per cent of the porosity within the implant is filled with woven and lamellar bone within 3 months. Moreover, the surfaces of Interpore-200 are intimately bonded with the bone tissue. The biomechanical properties of Interpore-200 blocks are similar to those of a cancellous bone graft. Once ingrown with vascularized bone tissue, the defect site is, in effect, restored. Interpore-200 adequately matches the elastic properties of bone so that stresses necessary to maintain healthy bone are transmitted throughout the regenerated region. Extensive animal and clinical studies have shown that nonporous implants or implants without interconnected porosity can result in aberrant mineralization, stress shielding, low fatigue strength, and bulk displacement. Hydroxyapatite with interconnected porosity like Interpore-200 reacts differently than materials with limited or no porosity. In animals, Interpore-200 exhibits 0 to 5 per cent biodegradation per year. Moreover, this minimal biodegradation is compensated by regeneration of bone. These studies have now been extended for 4 years. Interpore-200 and its ingrown bone are remodeled in response to the same chemical and biomechanical forces that remodel normal bone. Therefore, Interpore-200 responds in accordance to Wolff's law. Having achieved an optimal combination of biomaterial (hydroxyapatite) in an ideal porous structure (replamineform), Interpore-200 fulfills the expectation of early researchers in the basic sciences who demonstrated that an interconnected porous material is better tolerated by the body than the same material in solid form.(ABSTRACT TRUNCATED AT 400 WORDS)

A family of epidermoid lung cancer models.

A method of sustained release implantation has been developed whereby Silastic cylinders, impregnated with benzo[alpha]pyrene (BP) or methylcholanthrene (MCA) each at 2% (low dose) and 10% (high dose) concentrations, were inserted into the bronchus intermedius of hamsters. High-dose BP and MCA, and low-dose MCA had first-order exponential release rates: the half-time of release was 40 days for high-dose BP, 30 days for high-dose MCA, and 165 days for low-dose MCA. Release rate of low-dose BP was a second-order function: half-time of release was 40 days. Atypical squamous metaplasia was noted by 4 weeks in more than 65% of hamsters after insertion of each high-dose carcinogen but in less than 30% with the low-dose carcinogens. Carcinoma in situ was noted approximately 8 weeks after high-dose BP and 19 weeks after low-dose BP. At about 15 to 17 weeks after a high-dose carcinogen, 64% of animals had invasive epidermoid cancer, whereas after a low-dose carcinogen, only 21% did. After 25 weeks of exposure to a high-dose carcinogen, more than 85% of hamsters had invasive epidermoid cancer; up to 52 weeks were required for invasive epidermoid cancer to develop in 30% after a low-dose carcinogen. Measured by image analysis, nuclear deoxyribonucleic acid content of cells with severe atypical squamous metaplasia was greater than tetraploid (mean +/- standard deviation [SD], 3.77 +/- 1.4), whereas cells with invasive epidermoid cancer were suprahexaploid (mean +/- SD, 6.48 +/- 3.6). These differences are significant (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A clinically relevant canine lung cancer model.

Research on early human lung cancer is difficult; we have sought a canine correlate. Regimens included endobronchial submucosal injections and topical focal applications of benzo[a]pyrene, nitrosomethylurea, dimethylbenzanthracene, and methylcholanthrene, singly or in combinations. Sustained-release discs were placed into lung parenchyma or sutured into major bronchi. Tracheal segments were isolated as cervical pedicle grafts. Gross and histological evolution was reproducible. Columnar and basal hyperplasia and squamous metaplasia were early changes. Atypia occurred within 6 weeks and was found in all dogs within 16 to 18 weeks. Invasive cancers occurred within 8 to 65 months. No tracheal graft developed cancer. Of 15 dogs with parenchymal sustained-release implants, 1 to date has developed cancer in 8 months. Four endobronchial regimens have produced 16 cancers in 56 lungs at risk for 18 to 65 months. No cancers developed in 23 lungs at risk from eight other regimens. Of 10 dogs at risk for unilateral endobronchial cancer, 5 have had cancer. Of 23 dogs with both lungs at risk, 9 developed cancer. We have shown focal carcinogenesis with well-defined pathogenesis and an extended preneoplastic period at predictable sites in a lung cancer model.

Induction of lung cancers in preselected, localized sites in the dog.

Cancers were induced in 10 dogs in preselected sites within the lung. These 10 animals were among 89 dogs enrolled in 10 protocols with different carcinogens, doses, and techniques over 6 years. The 10 dogs that developed cancers were included in five of these protocols; 4 of the cancers were included in one of the protocols. The administered carcinogens included the aromatic hydrocarbons benzo[a]pyrene (BP), N-methyl-N-nitrosourea (MNU), 7, 12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), and yttrium-91 (91Y) beta radiation. The techniques of application included intrabronchial and intrapulmonary sustained-release implants of DMBA and 91Y, serial intrabronchial submucosal injections of BP, DMBA and MCA, or topical application of MNU. The intervals from the first application of a carcinogen to the diagnosis of malignant disease ranged from 8 to 56 months. Of the tumors induced, 9 were centrally located squamous cell carcinomas and 1 was a peripherally located carcinosarcoma. Four of the dogs were killed or died with stage 3 disease. The remaining 6 dogs are alive and are being evaluated serially; all 6 are in stage 1.

Sustained release of benzo(a)pyrene from silicone polymer into the tracheobronchial tree of hamsters and dogs.

A method was developed to expose specific sites of the hamster and canine tracheobronchial tree to benzo(a)pyrene (BP) at quantitatively sustained rates. Implants for sustained release were formed by incorporating BP in a silicone rubber matrix at concentrations of 9.05 to 12%. Forty-nine hamsters and 12 dogs had a total of 86 implants surgically adhered to the tracheobronchial mucosa for up to 200 days. BP was released from the implants in hamsters and dogs as a first-order exponential function with a half-time of 54.8 and 44.5 days, respectively. Pathogenesis was progressively time and dose dependent. Squamous metaplasia with atypia regularly occurred in dogs within 150 days or after 7.17 mg BP and in hamsters after 50 days or 288 microgram BP. Bronchogenic cancers occurred in 93% of our hamsters after 100 days and 467 microgram BP. This method has applicability potentially as a bioassay for evaluating carcinogens in hamsters and currently as a means of producing a model of lung cancer in which neoplasia is induced at precise, selected sites.

Cytologic correlates of chemical carcinogens in respiratory tracts of dogs.

During canine respiratory carcinogenesis studies with benzo[a]pyrene (BP) and N-methyl-N-nitrosourea (NMU), a stereotypic pattern of cytomorphology characteristic for each carcinogen was observed. In the early stage, BP induced changes primarily in the cytoplasm. These changes consisted of incresaed size, basophilia, and vacuolization. In contrast, NMU induced primarily nuclear enlargement and enhanced the prominence of the nucleolus. Subsequently, cells exposed to BP demonstrated pleomorphism of the cytoplasm and nucleus, whereas cells exposed to NMU formed bizarre spindle-shaped cells. Cells were generally more scattered from one another following BP treatment than after NMU treatment. When the mucosa was exposed to NMU after the previous use of BP, the cytologic findings promptly became those characteristic of NMU.

Sustained-release implants of chemical carcinogens in the canine tracheobronchial tree.

A proved carcinogen, benzo[a]pyrene (BP), was incorporated into liquid silicone rubber polymer which was then vulcanized into solid form. Discs of BP polymer containing 9.05 to 12.12% BP were sutured into the trachea and right main bronchi of 10 dogs which were observed for 3 to 8 months. The discs were either in contact with the endobronchial surface of the mucosa or placed into submucosal pockets. All discs that were implanted submucosally remain in place. Only 2 discs implanted by the fixation method were extruded. The pocket implants of BP polymer caused ulceration and erosion of the mucosa; these changes did not occur from discs of polymer without carcinogen. To date these implants have induced squamous metaplasia with atypical changes. A method for sustained-release implantation of carcinogens in the canine tracheobronchial tree has been developed.

Experimental canine tracheal grafts with reversible squamous metaplasia.

A technique for making bistomal tracheal pedicle grafts in dogs is described. The grafts were placed subcutaneously in the neck, and their mucosa was readily accessible to serial manipulations, endoscopic examinations, and biopsies, without requiring anesthesia. Squamous metaplasia developed in 5 of 8 grafts, or 63 per cent, within 1 week. Within 12 weeks, squamous metaplasia was seen in all grafts, and it was consistently accompanied by basal hyperplasia. After orthotopic reimplantation of the grafts, the squamous metaplasia proved to be reversible within 4 weeks. The preparation used for making these observations is a valuable research tool for sequential studies of the respiratory tract mucosa.