Both hyaluronic acid and CGRPa have been found to be stem cell mitogens, in vitro and in vivo. The hypothesis of the study is that both molecules aid in the process of bone formation around dental and orthopedic implants to ensure increased biointegration. Accordingly the present experimental animal study was developed to ascertain whether there are any enhancement effects during bone growth around Hydroxyapatite (HA) and Titanium (Ti)polycarbonate coated implants with the use of hyaluronic acid (Hyal act) and Calcitonin Gene Related Peptide a. (CGRPa ). For the purpose of this investigation two beagle dogs were used. Three different implants were implanted in the dog femurs, HA coated, Ti coated, and uncoated polycarbonate plugs . Each of the three type of implants was placed alone as controls. Fifteen implants had hyaluronic acid (two HA coated; two Ti coated and one Polycarbonate )or CGRPa (two HA coated; two Ti coated and two Polycarbonate ) or the combination of the two substrates (two HA coated; two Ti coated and one Polycarbonate) adsorbed to the implant surface(tests). Animals were sacrified at two months and block sections were taken. Semithin serial cross sections along the short axis, 5-6 micron thick, were taken from each implant and viewed under the light microscope. Digitized histomorphometric analysis was performed to quantitate the total bone area interfaced with the implant surfaces. Results showed a statistically significant increase of implant surface lined by bone in the HA coated implants with hyaluronic acid, with CGRPa and with the combination of hyaluronic acid and CGRPa when compared to the HA coated implants alone. The Ti coated implants with hyaluronic acid, with CGRPa and the combination of hyaluronic acid and CGRPa showed a statistically significant more bone apposition than Ti coated implants alone although less overall than the HA implants. The polycarbonate implants were between the HA and Ti implants in bone apposition. The conclusion of the study is that both hyaluronic acid and CGRPa are capable of enhancing bone growth around dental implants in the animal model.
Numerous studies have accelerated interest in the potential of growth factors to enhance and promote the healing of bone at the implant interfaces.
Reports in the oral implant literature during the last two decades suggest that bone can directly interface and contact cylindrical screw shaped titanium implants (Hansson et al.: 1983 and Sisk et al.: 1992), titanium alloy implants( Lum et al.: 1986 and 1988), titanium plasma spray implants ( Listgarten et al.: 1992,Schroeder et al.: 1981), porous titanium implants ( Deporter et al.: 1986, and Keller et al.; 1987 ), hydroxyapatite-coated titanium implants (Cook et al.: 1987, and De Lange et al.: 1989), titanium blade implants (Akawaga et al.: 1986), and single-crystal ceramic implants (Steflik et al.: 1992).
Quantification of the nature of this bone interface by Deporter (1986) et al. and Hipp and Brunski (1987) have suggested that approximately 50 to 65% of the implant surface is apposed by bone for cylindrical titanium and porous titanium implants.
To be accurate and not speculative characterization and description of the nature of the bone tissue response to dental implants must be accomplished with thin tissue section resolution. One of the ongoing problems of interpreting data from histological sections is that most of the research is derived from block sections of metal or metal coated with hydroxyapatite. Because of the metal, tissue sections, of necessity have been thick, ranging down from 150 microns to 10 microns (Donath et al. 1982).
Even so Hansson et al. (1983), Albrektsson et al. (1981, 1987), and Linder et al.(l983) have shown some equivocal evidence of close bone proximity to titanium implants using transmission electron microscopy. Furthermore they suggested that a 2-200 nm thick ground substance layer exists between bone and titanium implants. This ground substance consists of proteoglycans. These glucosaminoglycans consist of monosaccharides, including hexosamines, interconnected by glucosidic bonds. It has been suggested that these proteoglycans are important for the bonding, through their hydroxyl groups, to the titanium oxide present on the titanium implant surface (Albrektsson et al. 1983). The oxide layer is built up of a combination of Titanium oxides, the stable coating of about 50-2000 Angstroms after fixed prosthesis loading for 6 years. The oxide had incorporated phosphorus, calcium, and sulfur while implanted, and this was interpreted as an indication of interaction with the body tissue (McQueen et al. 1982) Recently Steflik et al. (1994) identified osteocytes both at a distance from the implant and closely situated to the bone-implant interface. In this same study there were densely mineralized thick collagen fibers apparently extending to within 100 nm of the implant surface. Interposed between the layer of mineralized thick collagen fibers and the implant surface was a zone of mineralized, more finely fibrillar material. The same authors also claim to demonstrate the presence of an osteocyte approximately 5 mm from the interface to the implant at the electron microscopic level. Also, in the same study, there is the demonstration of a thin not mineralized connective tissue layer separating the mineralized and the implant surface. However their histologic material was obtained by separating the whole implant, from the surrounding tissue, by immerging the block sections into liquid nitrogen followed immediately by immersion into boiling water In another study Serre et al. (1994) have shown a close contact between calcified bone tissue and titanium; also, a close contact was observed by newly formed osteoid tissue and the titanium implant surface. In some other sections the same authors demonstrated the presence of a seam of loose extracellular matrix separating the mineralized phase and the implant.
In 1963 Smith reported favorable results with ceramic implants. Since then, these materials have been widely used in surgery. Many reports are now available dealing with the clinical use of hydroxyapatite for jaw augmentation ( Kent et al. 1982), tooth replacement (Tracy et al. 1984, de Putter, 1986), bone substitution (Jarcho, M 1981), and middle ear reconstruction (van Blitterswijk et al. 1985). The potential advantage of hydroxyapatite implants is to produce an intermediate region between bone and implant that allows for a more intimate contact and bioactive integration with a direct bond between the mineralized tissue and the implant.
There are many hydroxyapatite studies demonstrating an intimate, direct bone contact without any visible interruption. de Lange et al. (1990) showed bone growing into irregularities of Ha coated implants. Calcified bone matrix is observed within a distance less than 500 A from the implant surface. They demonstrated a thin electron dense layer of organic material at the bone implant interface. This layer had a thickness of 20-100 Angstrom and had many similarities with the lamina limitans of bone. This organic bone layer was also observed at the inner walls of the osteocyte lacunae in bone tissue deposited on the implant surface. The direct bonding between bone and hydroxyapatite is shown by de Lange and Donath (1989 ), although in the same study they also demonstrate the presence of some interruptions of the HA coating on the implant surface. They identified some "ceramic particles embedded in the ingrown bone or lying in the cytoplasm of locally situated macrophages". They alluded to some possible biodegradation or resorption phenomenon rather than manufacturing problems to explain this particular finding.
Previous experimental studies by Golijanin and Bernard 1987, 1988, showed that in vitro ( Bernard et al. 1990, Holden et al. 1990, Bernard et al. 1993) as well in vivo (de Groot et al. 1987, Jarcho 1992) there is a higher percentage of contact and biointegration between bone and hydroxyapatite than between bone and titanium. Bernard et al. (1993) in a comparative animal study demonstrated that 25% of the interface between bone and hydroxyapatite coated implants were osseointegrated after 3 months demonstrated at the ultrastructural level. None of the titanium implants showed a direct bonding between mineralized tissue and the implant surface: rather there was always a thin seam of fibrous connective tissue interspersed between the bone and the implant. In another experimental trial, Gottlander et al.( 1992) demonstrated significantly more direct bone contact in the hydroxypatite coated specimens compared to the titanium plasma sprayed coated implants. A 75.9% of bone contact was observed for the hydroxyapatite versus a 59.9% of the titanium coated. In a different study Weinlaender et al . ( 1992) showed also a higher percentage of bone contact in the Ha coated specimens compared to titanium plasma sprayed and titanium screwed implants. They demonstrated a 45%, 55% and 71% in the titanium screwed, the plasma sprayed and in the Ha coated implants respectively.
Different results have been shown by Steflik (1996 ) who demonstated that 41% to 50% of the surface of ceramic implants were apposed by bone, whereas between 50% and 65 % of the surfaces of titanium implants were appposed by bone in an investigation of 120 endoosteal implants where the implants have been in place for up to 29 months and supported fixed partial dentures for periods up to 24 months.
Bone growth factors
It is well known that mineralized tissue has a remarkable degree of remodeling, repair and regeneration. The cellular and molecular basis of bone regeneration and remodeling were postulated and defined by Urist in 1965. He discovered that demineralized lyophilized bone segments were able to induce bone formation if inserted in rabbit muscles. From that time on, several studies have demonstrated the presence of " bone growth factors" which are able to characterize and induce bone formation. In 1982 Farley and Baylink reported that extracts of human bone contained abundant mitogenic activity for embryonic calvarial bone cells. In 1988, "skeletal bone growth factor", the most abundant growth factor stored in human bone was purified to homogeneity and was shown to be very similar if not identical to insulin like growth factor-II purified from human serum (Mohan et al .1988). During the purification of IGF-II from human bone extract, evidence was found for the presence of additional growth factors in human bone. Characterization of these additional bone growth factor activities revealed that human bone matrix contains multiple growth factors, including IGF-I, as well as IGF-II, transforming growth factor-b (TGF,b), platelet derived growth factor (PDGF), and basic fibroblast growth factor ( basic FGF), human bone matrix does not contain detectable amounts of epidermal growth factor, IGF-II and TGF,b are the two most abundant growth factors present in human bone matrix, and IGF-I, PDGF, and basic FGF are several fold less abundant ( Mohan et al. 1987).
The first isolation of bone morphogenetic proteins were initiated by Urist 1965, followed by Wozuey et al., 1988, Luyten et al., 1989; Celeste et al., 1990 Sampath et al. 1990 and Kinglsley et al. 1994 . The subsequent isolation of the genes encoding these proteins from human cDNA libraries identified a family of proteins including BMP-2 Through BMP-9 (Tables I, II, III ). The predicted amino acid sequences of BMPs indicate that they are all members of the TGF-8 superfamily, sharing a high degree of homology within the C-terminal cysteine domain ( Celeste et al., 1990; Luyten et al., 1989; Ozkaynak et al., 1990; Sampath et al., 1990) . TGF-8 oc is an abundant growth factor in bone matrix and has complex effects on a variety of bone cell types. In vitro, it has biphasic effects on bone cells and can increase or decrease proliferation. It substantially increases matrix synthesis by osteoblasts. TGF-B resides within the bone matrix in a latent form. Because it can be locally activated (Oreffo et al. 1989) and has effects on both bone-forming and bone-resorbing cells, it is a candidate molecule to be involved in the coupling mechanism of bone remodeling (Wozuey, 1995).
Growth factors have been found in dentin extracts and the concentrations of these growth factors were similar to those found for human bone (Finkelman et al. 1990). In addition, extracts of cementum have been shown to be mitogenic to gingival fibroblasts in culture (Miki et al. 1987). Thus growth factors are present in abundance not only in bone but also in other hard tissues, such as dentin and cementum.
With regard to the in vivo actions of BMPs, there have been several studies demonstrating that implantation of recombinant BMPs can induce bone formation (Wozney et al. 1988 Luyten et al. Celeste et l. 1990; 1989; Sampath et al. 1990; Wang et al. 1990;). In 1994 Cook et al. showed the effect of osteogenic protein - 1(also known as bone morphogenetic protein7) in a rabbit ulnar non-union model in large segmental osteoperiosteal defects. The defects treated with either human recombinant osteogenic protein or bovine osteogenic protein in combination with an allogenic-bone collagen carrier, revealed, in both cases, the capacity of forming new bone which had new cortices with advanced remodeling and marrow elements after a period of eight weeks. Controls, with the collagen carrier alone, showed no bone formation at any of the sites.
With regard to alveolar bone, great interest has been generated concerning the use of growth factors to restore and regenerate mineralized tissues lost as a result of human periodontal disease. Recent studies have reported that the use of a combination of PDGF and IGF-I was able to significantly enhance the formation of the periodontal attachment apparatus during the early phases of wound healing following surgery in the beagle dog and in the monkey ( Lynch et al. 1991, Rutheford et al. 1992) . In another animal study Becker et al. ( 1992) used PDGF/IGF-I with Guided Tissue regeneration techniques to augment bone formation around immediate extraction socket implants. More recently Sigurdsson et al.(1995) demonstrated the capacity of recombinant human BMP-2 to significantly regenerate the periodontium around teeth in the beagle dog model. Significant amounts of new cementum and new and denser alveolar bone resulted after 8 weeks of healing.
Calcitonin Gene Related Peptide (CGRP)
The existence of CGRP was postulated in 1983 by Rosenfeld who discovered the mRNA of CGRP from a calcitonin rat cancer. The first isolation of the peptide was completed by Morris et al. (1984) in studies of human medullary thyroid carcinoma. CGRP is a 37 amino acid peptide resulting from the specific maturation processes of calcitonin gene products. Its mRNA is closely related to calcitonin and amylin, and to lesser extent, to the region coding for the alpha chains of relaxing, insulin and insulin growth factors. In thyroid C cells, calcitonin is the major gene product, but CGRP is predominant in the central and peripheral nervous system. CGRP is found in most tissues and is considered to be a neuromediator of particular importance in the cardiovascular system.
The gene of calcitonin, Calc I is located on the short arm of chromosome 11 and it express two mRNAs; one for the precursors of calcitonin an the other for the codification of CGRPa . Then, another gene, Calc II, situated on the same chromosome, but in another region, expresses the mRNA for CGRPp which differs from CGRPa by three amino acids.
CGRP has been characterized as a neuroactive substance which is expressed in a large proportion of small to medium diameter sensory ganglion neurons whose central terminals lie in the superficial spinal and medullarydorsal horn. A large percentage of these neurons display CGRP-like (CGRP-LI) immunoreactivity in Raschkow's plexus and the odontoblastic layer of the dental pulp and into dentinal tubules of calcifying tooth structure (Silverman et al. 1987). Surgical deafferentation of the inferior alveolar nerve resulted in depletion of CGRP-LI, indicating the sensory nature of the CGRP-LI fibers. (Silverman et al. 1987). CGRP is a powerful endogenous vasodilator in man. A plasma concentration of 56 pmol/1 (slightly above physiological levels) provokes flush, hypotension and secondary catecholamine release with subsequent tachycardia. Intravenous injections lead to systemic vasodilatation and redistribution of blood flow to the skin, the brain, and probably the splanchnic territory. It has been postulated that CGRP plays a role in blood pressure regulation in certain pathological conditions (Bunker et al. 1990; Goadsby et al. 1990. Gennari et al., 1991;). It has also been related to cardiovascular effects which include positive chronotropic effect and inotropic action. Both centrally and periferally injected CGRP inhibits gastric secretion (Fisher et al. 1983; Tokami et al. 1985). The presence of CGRP has also been demonstrated in both endosteum and periosteum of long bones and periosteum of cranial bones ( Kruger et al. 1989). It has a parathyroid hormone (PTH)-like effect at high doses in the rabbit and in the chick. Paradoxically, at low doses in the rabbit, the peptide has a calcitonin (CT) like effect (Tipping et al. 1984; Struthers et al. 1985;).The CT-like effects of CGRP in the rabbit are due to a direct inhibition of osfeoclastic bone resorption ( Zaidi et al. 1988). It has been shown that cAMP production in human cells can be enhanced by CGRP by as much as 30 to 50 fold (Crawford et al. 1985 ). It was also demonstrated that chick, rat and mouse bones contain cells in an osteoblast-rich population that respond specifically to CGRP with a rise of cAMP ( Michelangeli et al. 1989). More recently, it was reported that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies in vitro and ex vivo. The same authors, in a subsequent study, showed the doserelated response of CGRP and its osteogenic potential(Bernard and Shih 1989,1990,1992). Furthermore, in another tissue intimately related to mineralization, Carnes and Harris in 1994 postulated that CGRP together with TGF-b1 is able to stimulate the production of BMP-2RNA in human pulp tissue cells.
Hyaluronic Acid
Hyaluronic Acid (HA) is a polysaccharide molecule composed of alternating units of D-glucuronic acid and N-acetyl D-glucosamine. HA assumes various sizes and shapes in solution, depending on its molecular weight ( Laurent, 1970). The hydroxyl groups on HA molecules allow HA to be readily hydrated and extensively dispersed, causing it to occupy an extremely large volume (Laurent, 1970). This "swelling" property of HA is functionally crucial for its biological role of stimulating cell migration and proliferation during early embryogenesis (Laurent, 1970; Kosher et al., 1981). In recent years, HA has been reported to play critical roles in a wide variety of biological events, such as wound healing, chondrogenesis, osteogenesis, the immune response, and migration of rat transformed cells (Kubler and Urist, 1990; Boskey et al., 1991; Lesley and Hyman, 1992; Noble et al., 1993; Turley et al., 1993;).
HA was found to be an aggregating factor in chick embryo and mammalian cell cultures ( Pessac, 1972). At low concentration, HA stimulated cell aggregation, and at high concentration, HA inhibited it (Pessac, 1972). In 1979, Toole showed that 3[H]-hyaluronate directly bound the surface of cultured SV3T3 cells. This binding was specific for HA molecules; structurally related molecules (e.g., dermatan) had little or no effect on HA binding in a competition assay, which suggested the existence of HA specific proteins (HABP) on the cell surface. The mechanism of HA-mediated cell aggregation and disaggregation was proposed based on intercellular crossbridging. Aggregation prevails at low HA concentration but is prevented at high concentration due to the saturation of HABPs (Toole, 1979). The ability of HA to inhibit or stimulate cell aggregation at high and low concentrations, respectively, correlates with the finding that in low concentration areas of HA along the proximodistal axis of the embryonic chick limb bud, there is established a distinct zone of cell proliferation, and of cytodifferentiation (Kosher, 1981). HA stimulates cell migration at high concentration by inhibiting intercellular aggregation, and at its reduced concentration it enhances cell aggregation and condensation, which leads to overt cytodifferentiation. Earlier studies point to the idea that HA may be involved in osteogenesis. Iwata and Urist (1973) showed that " implants of osteogenetic preparations of bone matrix accumulate HA at a significantly higher rate than implants of nonosteogenetic preparations". A recent genetic and biochemical study with brachymorphic mice has shown that matrix mineralization, which normally takes place in a high sulfate glycosaminoglycan environment, was rigorously enhanced in an undersulfated GAG environment (Boskey, 1991). Since HA is the only nonsulfated GAG, a higher percent content of HA in extracellular matrix might favor mineralization of the calcifying matrix.
Pilloni and Bernard (1994) have investigated the effects of HA on embryonic osteogenesis in vitro using a fetal mouse calvarial system. They showed that low molecular weight HA molecules (e.g., 30 kD and 40 kD) triggered the formation of increased number of osteogenic colonies as compared to the control group, while higher molecular weight HA molecules (e.g., 160 kD, and 1300 kD) had no effect. More recently Kang et al.(1995),using adult bone marrow instead of fetal mesenchyme, have shown that the greatest increase in the number and size of osteogenic colonies was seen with 160 kD at low concentration (i.e. 0.5 mg/ml) and they also have postulated that cell proliferation is enhanced by intercellular aggregation. These apparent differences in the results between Pilloni et al.(1994) versus Kang et al (1995), can be resolved with an assumption that the quantity of HA Binding protein per cell might be higher on the surface of the embryonic mesenchymal cells of the calvaria (Pilloni et al. 1994) than that of the osteogenic stem cells of the adult bone marrow (Kang et al.).There is support for this hypothesis. First, the extent of osteogenic stimulation by 30 kD and 40 kD HA was positively related to increased HA dosage in embryonic model (Pilloni and Bernard, 1994), while the extent of osteogenic stimulation by 160 kD HA was negatively related to the dosage of HA in the adult model. This indicates that cell surface bound HA Binding Protein (HABP) of bone marrow stem cells was saturated with HA at one concentration (e.g, 160 kD HA at 0.5 mg/ml), at which HABP of fetal mesenchyme was not completely saturated. That is, the embryonic mesenchyme may possess a higher quantity of the HABP per cell than does the adult stem cells. Second, earlier biochemical studies showed that the high molecular weight HA binds to cell surface with a greater binding affinity than does the lower molecular weight HA because a larger HA molecule can simultaneously bind to a greater number of HABP (Underhill and Toole, 1980). Conversely, it can be postulated that cells which express a higher quantity of HABP may interact with low molecular weight HA molecules as efficiently as do cells which possess the lower quantity of HABP when they interact with high molecular weight HA.
Most recently Sasaki and Watanabe (1995) showed a that Hyaluronic acid is capable of accelerating new bone formation through mesenchymal cell differentiation, in bone created wounds in the animal model. They were able to demonstrate that bone formation had already been induced at day 4 after the application of HA.
Research Plan: A total of 2 dogs were utilized. 10 implants were placed in animal 1 and 8 in animal 2. Both dogs were sacrificed at 8 weeks postoperatively. At sacrifice, the implants were removed in block sections and studied with light microscopy. Histomorphometric analysis was performed.
Animal Model: Two male beagle dogs were chosen because of their availability, ease of handling, anatomical size and bone repair remodeling characteristics. The animals were 1 year old and were initially screened to exclude acute and chronic medical conditions during a 2 week quarantine period prior to surgery. Specific attention was taken in selecting animals of uniform size and weight to limit the variability of implant fit and loading. Dogs were radiographically screened prior to surgery to rule out any preexisting pathology and to assure proper femoral size to accept the implants.
Implants:
Three types of implants were placed: All were 5mm x 12mm cylinders Figure 1.
Anesthesia procedures: The animals were pre-medicated with 20-25 mg of acepromazine administered IM. Anesthesia consisted of sodium pentobarbital (nembutal administered IV, 50 mgtml ).
Surgery: Using standard aseptic techniques, surgery was performed under anesthesia and was monitored by EKG and heart rate monitors. A record of animal surgery including its postoperative course was maintained for each animal.
A lateral incision was made from the greater trochanter proximally to the distal femur. Careful dissection of tissue and retraction of overlying tissues was performed to the cortex of the femur. Using a powered surgical drill and sequential drill bits with diameters beginning at 3.5 mm and ending with 5.5mm, the implantation sites were prepared.
The surgical defects were made in the metaphyseal, mid diaphyseal and distal third regions of the femur to accomodate the implants. During drilling the bits and the bone were constantly cooled with irrigation of physiologic saline solution delivered by syringe. The final implantation site was approximately 1 mm oversized. Then in order to achieve primary stabilization autologous bone chips were harvested and wedged around the cortical portion of the implants without penetrating the trabecular bone nor smearing the implant surface. Before insertions all implants except for A4, B4 and C4, were coated with a gel (lurocoat ophtalmic viscoelastic 1 ) solution of Hyaluronic acid or CGRPa: or a combination of the two (Table V) Figure 2, Figure 3.
The concentration of the solutions used were 0.5 mg/ml for the hyaluronic acid (mw 130-160kD) and 30 mg/ml for the CGRPa. Routine, closure and suturing followed.
Animals were administered IM antibiotics at the start of surgery. A course was maintained for each animal on a form customized for the proposed study. The animals were administered Butorphanol (0.025 gm/lb) for pain in the morning following the procedure and monitored as needed. Routine anterior-posterior Xrays were taken immediately after surgery to ensure proper surgical placement.
Postoperative: Animals were kept in recovery cages for 24h postoperatively after which they were transferred to standard dog runs for long term housing.
ObservationsClinical Examination: All animals were examined daily at approximately the same time by a qualified technician for mortality, clinical signs of ill, health, behavior changes, or adverse reactions to the implant. Particular attention was given to the surgical site.
Body Weight: Body weight was recorded once weekly prior to surgery and once weekly thereafter throughout the study and again prior to necropsy.
Terminal ProceduresGross Pathology: In order to avoid potential interferences from sistemic disease. a gross pathological examination of the animals organ systems was conducted immediately after death.
Animal Sacrifices: At the end of the study period, animals were sacrified using an IV barbiturate overdose. At the time of sacrifice 5 liters of Karnovsky's solution were used for intrarterial perfusion of the animals. The femurs were immediately harvested and specimens labeled. Sectioning and Histology: Upon sacrifice, intact femurs were retrieved and each implant site was isolated by transverse sectioning using a diamond saw. Each bone was removed in a block section. Postfixation of the specimens was performed in 1% tetraoxide of Osmium in water. Further ravages in distilled water preceeded the debydration step, which was carried out in serial alcohol solutions, 30-70-95-100%, each repeated for three times. Then, the specimens were diaphanized in Toluolo for three times, infiltrated with a combination of toluolo and Epon 812 and finally embedded in Epon.
The specimens were, then, sectioned longitudinally along the long axis of the implant in four quarters. Eight semi-serial sections 5-6 micron thick in cross section along the short circular axis were obtained from each quarter and stained with 1% toluidine blue in borax for light microscopy. All the semi-thin sections were taken in cortical bone. The amount of fibrous tissue and bone were determined using histomorphometrically computer software to establish the implant and surrounding tissue interface.
A total of 18 implants were placed in two beagle dogs. For each of the test implants, 8 sections were examined and a digitized histomorphometric analysis was performed. Four sections were analysed for each of the control implants.
Statistical Analysis . The mean percent bone values across the 12 different treatments were compared using one way analysis of variance ( ANOVA ) methods. The Tukey LSD ( Least Significance of difference) criterion was used to determine significance at alpha = 0.05 level. The individual Standard Error and pooled Standard Error were calculated.
The percentage of bone apposition is the amount of bone at the bone implant interface.
Table number VII displays the computerized histomorphometric data obtained with individual Standard Deviation for each unit, individual Standard Error and pooled SD. Data are presented as the percent (mean) of the implant surface apposed directly by bone. The Least Significant Difference LSD calculated is 24.6.
Hydroxvapatite coated implants:
The highest percent of bone apposition was for the HA coated implants with the addition of hyaluronic acid. ( 73.9%+5.3 SE).The HA coated implant with CGRPa alone and the combination of CGRPa and hyaluronic acid showed a bone apposition respectively of 61.6%+8.9 SE and 69.9%+5.1 SE. These results are significantly higher when compared to the HA coated implant alone (27.4%+10.4 SE) without the addition of the bioactive molecules. No statistically significant difference is present when comparing hyaluronic acid coating or CGRPa coating or the combination of the two Figure 4, Figure 5, Figure 6, Figure 7, and Figure 8.
Titanium coated implants:
The Ti coated implant with CGRPa had a mean 36.9% 11.9 SE of bone apposition. This is statistically significant compared with the Ti coated control implant(O%+O SE).
The Ti coated implants with hyaluronic acid and the combination of CGRPa and hyaluronic acid had a mean percent of bone apposition respectively of 9.4+7.0 SE and 8.3 %+6.7 SE. None of the sections for Titanium coated control implants have shown a direct bone contact to the implant surface (0%+0 SE bone apposition ). The difference between Ti coated with CGRPa is statistically significant when compared with Ti coated alone and Ti coated with hyaluronic acid and Ti with hyaluronic acid and CGRPa. Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 19.
Polycarbonate Implants:
The polycarbonate implants without coating demonstrated a mean of 11.9%+1.9 SE compared with 51.9%+11.8 SE bone apposition with CGRPa and hyaluronic acid, 47.9%+12.4 SE with hyaluronic acid, 47.9%+12.4 SE with hyaluronic acid alone and 45.7%+8.1SE with CGRPa alone. This was statistically significant when compared to the polycarbonate control implant without the use of any substrate. The inter-substrates differences are not statistically significant Figure 9, Figure 10, Figure 11, Figure 12, Figure 13.
The mean percent bone apposition around the HA coated implant was always statistically higher when compared to the mean percent of bone apposition around the Ti coated implant for all the tested biomolecules as well as without the added substrates. The polycarbonate implant bone apposition for all the different variables was higher than the Ti coated implants but lower than the hydroxyapatite coated implants.
During the past decade an increasing amount of studies have emphasized the concept that growth, form, and function of cells are modulated by highly specific interactions between the cells and their extracellular matrices and by growth factors. Most of such research suggests that specific cellular interactions are important not only for their growth but also for their morphology and function (Toole, 1973). These studies show that extracellular matrices are cell- and tissue specific, and progress has been made in characterizing these components and their specific functions. This is also the case of calcified tissues, because: "...Mineralized tissues are not simply the end result of precipitations of inorganic crystals from supersaturated solution in microenvironments that happen to be composed of organic molecules. They are, in fact, organic matrix-induced processes under the constant control of living cells..." (Fisher and Termine, 1985).
Hyaluronic acid possesses biochemical and physical properties suitable to perform an important role in the early events of osteogenesis as well as in many other tissues.
Hyaluronic acid is a prominent extracellular matrix component during bone morphogenesis (Toole and Gross, 1971) and large amounts of hyaluronic acid are present during the transition from mesenchymal cell to cartilage (Handley and Lowther, 1976).
In terms of its correlations with wound healing mechanisms and hard tissue development (Hay, 1980), hyaluronic acid can be thought as a "primer" in cell regeneration. A well defined sequence of events, already postulated by Weigel (1986), will better clarify this concept:
a) First, a matrix rich in hyaluronic acid is laid down in a cell-poor space. The property of hyaluronic acid of occupying large spaces with little mass functions as a space mantainer. Under these conditions, the highly replicating cellular elements, such as the osteoprogenitor stem cells were given the necessary extracellular space for the production of the daughter elements, which are formed at logarithmic pace.
b) Second, mesenchymal cell migration is stimulated and the hyaluronic acid matrix is infiltrated by cells migrating from the adjacent tissues: Cellular migration, as pointed out by Bertolami (1990), is a calcium-dependent mechanism. The highly charged molecule of hyaluronic acid causes the hydration of the ECM (Extra-Cellular Matrix) and consequent attraction of ions such as calcium.
c) Finally, cells within the hyaluronic acid matrix secrete both hyaluronidase, which degrades the hyaluronic acid, and sulfated glycosaminoglycans and collagen, which concomitantly replace some of the hyaluronic acid as the matrix is remodeled. Cellular elements have now the necessary amount of hyaluronic acid for the formation of the hyaluronic acid-dependent "cross-bridging". This concept is derived from findings on hyaluronic acid in cell regulation. Toole (1991) postulates the presence of large hyaluronic acid-dependent "pericellular coats" during the initial formation of cartilage in early development of the limb. He also proposes the above mentioned multivalent cross-bridging by hyaluronic acid of hyaluronic acid receptor sites on adjacent cells as a mechanism for cell aggregation. The two mechanisms together, pericellular coats and cross-bridging, lead to the formulation of the following hypothesis:
Soon after the removal of high levels of indigenous hyaluronic acid by the cellular hyaluronidase, cells-coats are created, in order to maintain space between them and to inhibit cell adhesion, so that they can keep migrating and dividing. Once this is accomplished, the degradation of hyaluronic acid by hyaluronidase allows the aggregation phenomenon of multivalent cross-bridging by hyaluronic acid to hyaluronic acid-receptor sites to occur.
Each of these three developmental systems (matrix rich in hyaluronic acid is laid down in a cell-poor space, stimulation of mesenchymal cell migration and enzyme secretion such as hyaluronidase) was part of the early hypothesis of Weigel (1986) about hyaluronic acid synthesis and degradation during early stages of tissue repair.
It is, then, very likely, that in the early stages there is the formation of a cell-poor hyaluronic acid matrix which is required before the complicated series of cell-mediated events that follows its destruction can procede. Therefor, cells in the process of replicating themselves need enough space. They have to avoid any cell-to-cell contact inhibition. As far as cell mediated events are concerned, a number of cell surface binding sites for hyaluronic acid have been reported on other cell types, such as endothelial cells and fibroblasts (Eriksson et al., 1983; Raja and Weigel; Underhill and Toole,1979). Hyaluronic acid is able of interacting both with mesenchymal stem cell surface and the other molecules in the extracellualr matrix. The versatility of hyaluronic acid, as most of the other proteoglycans, and its capacities for multiple interactions with other molecules gives it the ability to function as a multipurpose glue (Ruoshlati,1989) in cellular interactions. In fact, it has been suggested that a proteoglycan-mediated insolubilization of growth factors exists which provides a means of concentrating growth factors activity and directing them into a geometry appropriate for the architecture of the tissue. This has been demonstrated with heparin on Transforming factor beta and Fibroblast Growth factor activities (Castellot et al., and Vlodavsky et al. 1987).
Bernard and Shih (1990), demonstrated that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies. An increase in the number of bone colonies is probably caused by stimulating bone stem cell mitosis. The increase in size of the bone colonies was attributed to the stimulation of osteoprogenitor cell differentiation and/or osteoblast activity.
It has also been postulated by preliminary studies by Carnes et al.(1994) that CGRPa could be one step before the expression of BMPs. They showed that stimulation of pulp tissue cells with either CGRP or TGF-B1 resulted in increased BMP-2 mRNA.
"The analysis of bone and associated oral tissues comprising the support complex for endosteal dental implants is critical for understanding the mechanism for continued implant serviceability" ( Steflik 1994).The evaluation of the quantity of bone apposition directly apposed on the implant surface needs to be determined by the means of a histomorphometric analysis. "Osseointegration never occurs on 100% of the implant surface. Under the light microscope, bone appears to be in direct contact with the implant. By virtue of its excellent regenerative potential, bone is seen to grow around the ridges and grooves of screw-type implants and through the openings of blade and hollow cylinder-type implants. Remodeling of bone occurs constantly as part of the normal physiology of bone and continues after implant has been placed. Histologic sections of the bone-implant interface, however, are usually thick (20 to 150 mm ) and do not permit an accurate view of the interface. These thick sections have been the primary standard of viewing the interface and may have led to the premature definition of the interface as an osteointegrative one"( Bernard et al. in Carranza and Newman; 1996). In a previous study, Falez et al.(1994) used polycarbonate HA coated, Ti coated and grit blasted implants in order to obtain semithin sections of an approximate thickness of 5-6 ,mm and assess a more accurate interface between the implant surface and the sorrounding tissues. For the purpose of this investigation the same animal model and test device were utilized.
The results of this investigation illustrate that the addition of hyaluronic acid or CGRPa significantly enhanced the percentage of bone apposition at the implant surface. There was not any additional effect when the two active biomolecules were combined together. We can speculate that this lack of a summation effect is that they can be present at different steps in the osteogenic cells differentiation cascade. Both might act as mitogens but chronologically, separately, and probably activating two different mechanisms of the sequence of differentiation. The administration of the combination of both substrates could be not able to activate the two mechanisms at the same time. A possible explanation could be a negative bio-feedback type of relationship between hyaluronic acid and CGRPa.
A second possible explanation of the lack of additional effect, in bone formation, is the spatial conformation of hyaluronic acid and its charge density which might prevent CGRPa from directly stimulating the mesenchymal stem cells. According to the hypothesis postulated by Weigel in 1986, hyaluronic acid could play a role in mesenchymal cell differentiation at earlier stages, while CGRPa might come into the process later. A process of degradation might occur when CGRPa administered at the same time with hyaluronic acid, before it could produce any biologically significant effects. Further investigations need to be carried out to assess the different sequential action in the cascade of differentiation of bone growth.
However, a significant difference in bone apposition resulted after the use of CGRPa alone around Ti coated polycarbonate implants. The percentage of bone-implant direct interface was significantly higher (ANOVA statistical analysis) when compared to Hyaluronic acid alone and to the combination of Hyaluronic acid and CGRPa. Still, the bone apposition was less when compared to the HA coated implants. However, it has to be reported that a high individual Standard Deviation was calculated for the Ti coated implants with CGRP a alone 36.9% +32.9.
The dosage of both biomolecules was determined according to previous in vitro and ³ex vivi² investigations (Pilloni and Bvernard 1992, Kang et al. in press, Bernard and Shih, 1992). Evaluation of possible systemic effects was not assessed except for the clinical evaluation of changes of animal behavior for the duration of the expirament. No attempts were done to measure the duration of pharmacologic effects, the half life and the diffusion into the blood stream of both bioactive molecules. A more accurate systemic monitoring of all physiologic functions should be carried out, to rule out any possible undesirable side effects derived from localized use of the two substrates. Because the dimension of the relatively small peptide and the well-known effects on other systems, CGRPa could interfere with the normal physiology of other systems of any organism. On the contrary the dimensions of the molecule of hyaluronic acid and its ubiquitous presence in the extracellular matrix of the human and animal organisms suggest a minimum risk of long term side effects. Eventually, a specific biocompatible carrier system should be developed in order to locally deliver any bioactive substrate and prevent its diffusion into the systemic circulation.
The results of this experimental study showed a statistically significant increase of implant surface lined by bone in the HA coated implants with hyaluronic acid, with CGRPa and with the combination of hyaluronic acid and CGRP a when compared to the HA coated implants alone. The Ti coated implants with hyaluronic acid, with CGRP and the combination of hyaluronic acid and CGRPa showed a statistically significant more bone apposition than Ti coated implants alone although less overall than the HA implants. The polycarbonate implants were between the HA and Ti implants in bone apposition. The conclusion of the study is that both hyaluronic acid and CGRPa are capable of enhancing bone growth around dental implants in the animal model. Further investigations have to be carried out to evaluate the potential clinical applicability on the human model.
Akawaga Y, Hashimoto M, Kando N, Satomi K, Takata T, Tasuru H. Initial bone implant interfaces of submergible and supramergible endosseous single crystal sapphire implants. J Prosthet Dent 1986;55(1):96-99.
Albrektsson T, Branemark P-I, Hansson HA, Lindstrom J. Osseointegrated titanium implants. Acta Orthop Scand 1981;52: 155-170.
Al brektsson T, Branemark P- I , Han sson HA, Kas emo B , Lars son K, Lun dstrom I , McQueen DH Skalak R. The interface zone of inorganic implants in vivo: titanium implants in bone. Annals Biomed Engineering 1983; 11: 1-27.
Albrektsson T, Jacobson M.. Bone. Metal interface in osseointegration Prosthetic Dentistry 1987; 57(5):597-607.
Becker W, Lynch SE, Lekholm U, Becker BE, Caffesse, Donath K, Sanchez R. A comparison of ePTFE membranes alone or in combination with Platelet-deriveo growth factors and Insulin-like growth factor-I or Demineralized freeze-dried bone in promoting bone formation around immediate extraction socket implants. J Periodontol 1992;63:929-937.
Bernard GW, Golijanin LM, Holden C. The ultMstructural interface of titanium and porous hydroxyapatite with developing bone in vitro. UCLA Symposium on implants, Palm Springs,CA.1990.
Bernard GW, Falez P, Perugia L, Pilloni A, Gruber H. Qualitative and quantitative analysis of the bone implant interface, modern trends and perspectives in total hip implants, Roma ( Italy) May 13-15,1993.
Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. J Dent Res 1989; 67: 967.
Bernard GW, Shih C. The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide. Peptides 1990;11:625-632.
Bernard GW, Shih C. The enhancement of osteogenesis with neuropeptides in vitro and ex vivo. Biological mechanisms of tooth movement and craniofacial adaptation Ohio State University Press 1992: 475-484.
Boskey AL, Birgit LD. Hyaluronan interactions with hydroxyapatite do not alter in vitro hydroxyapatite crystal proliferation and growth. Matrix 1991;11:442-446.
Cook SD, Kay JF, Thomas KA, Jarcho M. Interface mechanics and histology of titanium and hydroxylapatite-coated titanium for dental implant applications. Int J Oral Maxillofac Implants 1987; 2(1):15-22.
Cook SD, Baffes GC, Wolfe MW, Sampath TK, Rueger DC, Whitecloud TS. The effect of recombinant human osteogenic protein-1 on healing of large segmental bone defects. J Bone Joint Surg 1994;76-A:827-838.
Crawford A, Evans DB, Sklodt H, Beresford JN, MacIntyre I, Russel RGG. Effects of human calcitonin gene-related peptide on human bone-derived cells in culture. Bone 1985;7:157.
de Groot K, Geesink R, Klein CPAT, Serekian P. Plasma sprayed coatings of hydroxylapatite. J Biomed Mater Res 1987;21:1375-1381.
de Lange GL, Donath K. Interface between bone tissue and implants of solid hydroxyapatite or hydroxyapatite coated titanium implants. Biomaterials 1989;10:121-125.
de Lange GL, de Putter C., De Wijs FLIA. Histological and ultrastructural appearance of hydroxyapatite-bone interface. J Biomed Mat Res 1990;24:829-845
Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S, Baylink DJ. Quantitation of growth factors IGF-I,SGF/IGF-II, and TGF-beta in human dentin. J Bone Min Res 1990;5:717-723.
Fisher LA, Kikkawa DO, RiverJE, Amara SG, Evans R, Rosenfeld MG, Vale WW, Brown MR. Simulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature 1983; 305:534.
Fisher LW Termine JD. Noncollagenous proteins influencing the local mechanisms of calcification Clin Orthop Rel Res 1985; 200:362-385.
Golijanin LM, Bernard GW. In vitro osteogenesis at the implant material-tissue interface. Proceedings 2nd Intern Congress in Preprosthethic Surgery, Palm Springs CA,1987: p. 4.
Golijanin LM, Bernard GW. Biocompatibility of implant metals in bone tissue culture. J Dent Res 1988;67:abs367.
Gottlander M, Albrektsson T, Carlsson LV. A histomorphometric study of unthreaded hydroxyapatite-coated and titanium-coated implants in rabbit bone. Int J Maxillofac Implants 1992;7:485-490.
Handley CJ, Lowther DA. Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in culture Biochim Biophys Acta 1976; 444, 69-74.
Hansson HA,Albrektsson T,Branemark P-I. Structural aspects of the interface between tissue and titanium implants. J Prosthet Dent 1983;50(1);109-113.
Hay ED. Development of the vertebrate cornea. Int Rev Cytol 1980; 63, 263.
Hipp JA, Brunski JB. Investigation of osseointegration by histomorphometric analysis of fixture bone interface. J Dent Res 1987; 66 (special issue):186.
Holden C, Bernard GW. Ultrastructural in vitro characterization of porous hydroxyapatitelbone cell interface. J Oral Implantol 1990;16:86-95.
Iwata H, Urist MR. Hyaluronic acid production and removal during bone morphogenesis in implants of bone matrix in rats. Clin Orthop Relat Res 1973;90:236-244.
Jarcho M. Calcium phosphate ceramic as hard tissue prosthetics. Clin Orthop 1981;157: 259-278.
Jarcho M. Retrospective analysis of hydroxyapatite development for implant applications. Dental Clinics North Amer 1992; 36:(1) 19-26.
Kang MK, Sison J, Bernard GW. Osteogenic stimulating effects of Hyaluronic acid on adult bone formation in vitro: influence of molecular size and concentration. 1995 In Press.
Keller JC, Young FA, Natiella JR. Quantitative bone remodeling resulting from the use of porous dental implants. J Biomed Mater Res 1987; 21:305-319.
Kent JN, Zide MF, Jarcho MJ, Quinn JH, Finger IM, Rothstein SS. Correction of alveolar ridge deficiencies with non resorbable hydroxyapatite. JADA 1982; 105:993-997.
Kingsley DM. The TGF,8 superfamily: New members, new receptors, and new genetic tests of function in different organisms. Genes Dev 1994;8:133-146
Kubler N, Urist MR. Morphogenetic protein-mediated interaction of periosteum and diaphysis. Citric acid and other factors influencing the generation of parosteal bone. Clin Orthop Relat Res 1990; 258:279-294.
Kosher RA, Savage MP, Walker KH. A gradation of hyaluronate accumulation along the proximodistal axis of the embryonic chick limb bud. J Embryol.exp. Morph 1981; 63: 85-98.
Kruger L, Silverman JD, Mantyh PW, Sterini C, Brecha NC. Peripheral patterns of Calcitonin Gene Related Peptide, general somatic sensory innervation: cutaneous and deep terminations J Comp Neurol 1989; 280: 291-330.
Laurent TC, Fraser JRE. The properties and turnover of hyaluronan. In: Functions of the proteoglycans, ed. by Evered D and Whelan, J Wiley Chichester, Great Britain, Ciba Foundation Symposium 124 1970; 9-29.
Lesley J, Hyman R. CD44 can be activated to function as an hyaluronic acid receptor in normal murine T cells. European Journal of Immunology 1992; 22(10):2719-2733.
Linder L, Albrektsson T, Branemark P-I, Hansson HA. Electron microscopic analysis of the bone-titanium interface. Acta Orthop Scand 1983; 54: 45-52.
Listgarten MA, Buser D, Steinemann SG, Donath K,Lang NP, Weber HP. Light and transmission electron microscopy of the intact interfaces between nonsubmerged titanium-coated epoxy resin implants and bone or gingiva. J Dent Res 1992;71(2) 364-371.
Lum LB, Beirne OR. Viability of the retained bone core in the Core-Vent dental implant. J Oral Maxillofac Surg 1986;44:341-345.
Lum LB, Beirne OR, Dillinges M, Curtis TA. Osseointegration of two types of implants in non human primates. J Prosthet Dent 1988;60:700-705.
Luyten FP, Cunningham NS, Ma S, Muthukumaran N, Hammonds RG, Nevins WB, Wood WI, Reddi AH. Purification and partial aminoacid sequence of osteogenin, a protein initiating bone differentiation. J Biol Chem 1989;264:1337713380.
Lynch SE, De Castilla GR, Williams RC, Christopher PK, Howell H, Reddy MS, Antoniades HN. The effects of short-term application of a combination of Platelet Derived and Insulin -like growth factors on periodontal wound healing. J Periodontol 1991;62:458-467.
Lynch SE, Buser D, Hernandez RA, Weber HP, Stich H, Fox CH, Williams RC. Effects of the Platelet derived growth factor/Insulin-like growth factor-I combination on bone regeneration around titanium dental implants. Results of a pilot study in beagle dog. J Periodontol 1991;62:710-716.
McQueen D, Sundgren JE, Ivarsson B, Lundstrom I, afEkenstam B, Svensson A, Branemark P-I, Albrektsson T. Auger electron spectroscopic studies of titanium implants. Adv Biomaterials 1982;4: 179-184.
Michelangeli VP. Effects of calcitonin gene-related peptide on cyclic AMP formation in chicken, rat and mouse bone cells. J Bone Min Res 1989;4(2):269272.
Miki Y, Narayanan AS, Page RC. Mitogenic activity of cementum components on gingival fibroblasts J Dent Res 1987;66:1399-1403.
Mohan S, Linkhart TA, Jennings JC, Baylink DJ. Identification and quantification of four distinct growth factors stored in human bone matrix. J Bone Min Res 1987; 2:44-47.
Mohan S, Jennings JC, Linkhart TA, Baylink DJ. Primary structure of human growth factor. Homology with human insulin-like growth factor-II.Biochim Byophys Acta 1988; 966:44-45.
Morris HR, Panico M, Etienne t, Tippins J, Girgis SI, Mac Intyre I.Isolation and characterization of human calcitonin gene-related peptide. Nature 1984;304:746748.
Noble PW, Lake FR, Henson PM, Riches DW. Hyaluronate activation of CD44 induces insulin-like growth factor factor-I expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages. J Clin Investig 1993;91(6) :2368-2377.
Oreffo ROC, Mundy GR, Seyedin SM, Bonewald LF. Activation of the bone -derived latent TGF beta complex by isolated osteoclasts. Biochem Biophys Res Commun 1989;158:817-823.
Ozkaynak E Rueger DC, Drier EA, Corbett C, Ridge RJ, Sampath TK, Oppermann H. OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J 1990;9: 2085-2093.
Pessac B, Defendi V. Cell aggregation: role of acid mucopolysaccharides. Science 1972; 175:898-900.
Pilloni A, Bernard GW. The effects of hyaluronic acid on intramembranous osteogenesis in vitro. J Dent Res 1992; 71:574 (IADR abstract t#471).
Raja RH, Weigel PH. Binding and internalization of hyaluronate by isolated rat liver cells. J Cell Biol 1985; 101, 426a.
Reddi AH, Cunningham NS. Initiation and promotion of bone differentiation by bone morphogenetic proteins. J Bone Miner Res 1993;8(2): 499-502.
Rollins BJ, Culp LA. Glycosaminoglycans in the substrate adhesion sites of normal and virus-transformed murine cells. Biochemistry.1979; 18: 141-148.
Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, River J, Vale WW, Evans RW. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 1983;304:129-135.
Ruoslahti E. Proteoglycans in cell regulation. The J. Biol. Chem..1989; Vol.264, 23: 13369-13372.
Rutheford RB, Niekrash CE, Kennedy JE, Charette MJ. Platelet derived and insulin-like growth factors stimulate regeneration of periodontal attachment in monkeys. J Periodontol Res 1992;27:285-290.
Sampath TK, Coughlin JE, Whetstone RM, Banach D, Corbett C, Ridge RJ, Ozkaynak E, Oppermann H, Rueger DC. Bovine osteogenic protein is composed of dimers of OP-1 and BMP-2A, two members of the transforming growth factorbeta superfamily. J Biol Chem 1990; 265:13198-13205.
Sasaki T and Watanabe C. Stimulation of osteoinduction in bone wound healing by high-molecular Hyaluronic acid. Bone 1995; 16:9-15.
Schroeder A, van der Zypen E, Stich H, Sutter F. The reaction of bone connective tissue and epithelium to endosteal implants with titanium-sprayed surfaces. J Maxillofac Surg 1981;9(1):15-25.
Serre CM, Boivin G, Obrant KJ, Linder L. Osseointegration of titanium implants in the tibia. Electron microscopy of biopsies from 4 patients. Acta Orthop Scand 1994;65(3):323-327.
Sigurdsson TJ, Lee MB, Kubota K, Turek TJ, Wozney JM, Wikesjo UME. Periodontal repair in dogs: recombinant human bone morphogenetic protein-2 significantly enhances periodontal regeneration. J Periodontol 1995;66:131-138.
Silverman JD, Kruger L. An interpretation of dental innervation based upon the pattern of calcitonin gene-related peptide (CGRP) immunoreactive thin sensory axons. Somatosens Res 1987;5(2):157-175.
Smith L. Ceramic-plastic material as a bone substitute. Arch Surg;1963; 87:653661.
Sisk AL, Steflik DE, Parr GR, Hanes PJ. A light and electron microscopic comparison of osseointegration of six implant types. J Oral Maxillofac Surg 1992;50:709-716.
Steflik DE, Sisk AL, Parr GR, Hanes PJ, Lake FT, Brewer P. Correlative transmission electron microscopic and scanning electron microscopic observations of the tissues supporting endosteal blade implants. J Oral Implantol 1992;18(2):110-120.
Steflik DE, Sisk AL, Parr GR, Lake FT, Hanes PJ, Berkery DJ Breer P. Transmission electron and high voltage electron microscopy of osteocyte cellular processes extending to the dental implant surface. J Biomed Mat Res 1994; 28:1095-1107.
Steflik DA, Lake TF, Sisk AL, Parr GR, Hanes PJ, Davis HC, Adams MO, Yavari J. A comparative investigation in dogs: 2-year Morphometric results of dental implant-bone interface. Int J Maxillofac Implants 1996;11:15-25.
Struthers AD, Brown MJ, Beacham JL, Morris HL, MacIntyre I. The acute effect of human calcitonin gene-related peptide in man J Endocrinol 1985;107s:129.
Takami, Kawai Y, Uchida S, Tohyama M, Shiotai Y, Yoshida H, Emson PC, Girgis S, Hillyard CJ, Mac Intyre I. Effect of calcitonin gene-related peptide on contraction of striated muscle in the mouse. Neurosci Lett 1985;60:227-230.
Tippins JR, Morris HR, Panico M, Etienne P, Bevis P, Girgis S; MacIntyre I, Azria M, Attinger M. The myotropic and plasma calcium-modulating effects of calcitonin gene-related peptide. Neuropeptides 1984;4: 425-434.
Toole BP. Hyaluronate and Hyaluronidase in morphogenesis and differentiation. Amer. Zool 1973; 13, 1061.
Toole BP, Gross J. The extracellular matrix of the regenerating newt limb: synthesis and removal of hyaluronate prior to differentiation. Developmental Biology 1979; 25:57-77.
Toole BP. Cell Biology of extracellular matrix, ed.Elizabeth D., New York: Plenum Press. 1991.
Tracy BM, Doremus RH. Direct electron microscopy studies of the bone hydroxylapatite interface. J Biomed Mater Res. 1984; 18:719-726.
Turley EA, Belch AJ, Poppema S, Pilarshi LM. Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B Lymphocytes . Blood 1993;81(2):446-453.
Underhill CB, Toole BP. Binding of hyaluronate to the surface of cultured cells. Cell Biol. 1979; 82, 475-484.
Underhill CB, Toole BP. Physical characteristics of hyaluronate binding to the surface of simian virus 40-transformed 3T3 cells. J Biolog Chem 1980; 255:45444549.
Urist MR . Bone: Formation by autoinduction. Science 1965;150:893-899.
van Blitterswijk CA, Grote JJ, Kuijpers W, Blok-van-Hoek CJG, Daems WT. Bioreactions at the tissue hydroxyapatite interface. Biomaterials 1986;6:243-245.
Vlodavsky I, Folkman J, Sullivan R, Fridman R, Ishai-Michaeli R, Sasse J, Klagsbru M. Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix. Proc Natl Acad Sci U.S.A.1987; 84: 2292-2296.
Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, HewickRM, Kerns KM, LaPan P, Luxemberg DP, McQuaid D, Moutsatsos IK, Nove J, Wozney JM. Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990;87:2220-2224.
Weigel PH, Fuller GM, Le Boeuf RD. A model for the role of the hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing. J Theor Biol.1986; 119: 219-234.
Weinlaender M, Kenney EB, Lekovic V, Beumer J, Moy PK, Lewis S. Histomorphometry of bone apposition around three types of endosseous dental implants. Int J Maxillofac Implants 1992;7:491-496.
Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA. Novel regulators of bone formation: Molecular clones and activities. Science 1988;242:1528-1534.
Wozney JM. The potential role of bone morphogenetic proteins in periodontal reconstruction. J Periodontol 1995;66:506-510.
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