Effects of fibrin on the integration hydroxyapatite coating implants: experimental study in a rabbit model

Experimental Animal Science

Effects of fibrin on the integration hydroxyapatite coating implants: experimental study in a rabbit model M. MORALES 1, R. NAVARRO2'3, M. ALMENARA2, J. M. MEDINA2, C. MELIAN 1 and C. GUTIERREZ 1 1Department of Animal Medicine and Surgery, University of Las Palmas de Gran Canaria, Arucas, Las Palmas, Canary Islands, Spain 2Department of Medical and Surgical Sciences, University of Las Palmas de Gran Canaria, Las Palmas, Canary Islands, Spain 3Orthopaedics Surgery and Tranmatology Service, Insular University Hospital of Gran Canaria, Las Palmas, Canary Islands, Spain

Summary The purpose of this study was to investigate the effects of the addition of fibrin (SAF) to titanium alloy implants coated with hydroxyapatite (HAP) on osteogenesis in rabbits. A titanium (Ti) alloy implant was inserted into the femoral neck of twenty-four adult rabbits. Six rabbits were included on each of the following groups: Ti control, HAP-coated Ti module, HAP-coated Ti module with added fibrin glue and Ti module also with added fibrin glue. After seven weeks, bone growth was examined radiographically and by histo-morphometry. The SAF/HAP mixture did caused to a significant increase in bone growth compared to the other groups. The addition of fibrin did not result in an increase in new-bone growth and increase the formation of fibroustissue in contact with the implant. We concluded that SAF did not demonstrate osteoinductive properties.

Key words: Hydroxyapatite, fibrin, rabbit model, coatting implants.

Introduction Bone ingrowth in non-cemented implants takes place in humans and small animals. However, analysis of retrieved implants has shown great variation in the degree of bone growth, varying in porous coatings from 10% (COLLIER et al., 1988) to 50% (PIDHORZ et al., 1993). Thus, irrespective of the influence of certain variables (implant design, age, metabolic state of the bone, etc.), research into biological fixation seems to be focussing on a number of agents which, when added to the surface of the implant, favour bone

J. Exp. Anim. Sci. 2002; 42:102-112 Urban & Fischer Verlag http://www.urbanfischer.de/journals/j eansc 0939-8600/02/42/02-102 $15.00/0

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growth and the speed with which it occurs, since this has a beneficial effect on the longevity of the prosthesis (ENGH et al., 1987; KEAVENYand BARTEL, 1995). Hydroxyapatite (HAP) ceramic coatings are bioactive coatings which have demonstrated their ability to increase the development and the extension of bone (GEESINKet al., 1987) in non-cemented endoprostheses, achieving a strong bond in a relatively short period of time (GEESINK et al., 1987; SOBALLE et al., 1993), even under load (SOBALLE et al., 1990). The addition of certain substances to the HAP in order to increase osteoblastic activity and promote osteogenesis is currently under study (ONO et al., 1990; SATO et al., 1991; NAKAMURAet al., 1998). Following in this direction, the angiogenic and fibrogenic activity shown by the fibrin adhesive (SAF - Tissucol Inmuno ®) may indicate the ability to increase osteoinduction. However, the influence of SAF as stimulant to osteogenesis is subject to debate. Some authors have found a beneficial effect of fibrin in wound repair (Albrektsson et al., 1982), in the repair of bone defects in Plastic and Reconstructive maxillofacial surgery in humans (BONUCCI et al., 1997; FORTUNATO et al., 1997), in the fixation of osteochondral fractures in dog femurs (KELLERet al., 1985), and in accelerating the repair process and new bone formation in bone defects of the proximal metaphysis of rats (ONO et al., 1990) and rabbits (SATo et al., 1991; NAKAMURAet al., 1998). Also, Ig~ANIAet al. (1998) reported benefitious effects when fibrin was added to coral implants compared to coral alone group in an experimental study carried out in rabbits. However, other authors have not found any stimulatory effect of fibrin and have observed a higher soft tissue development with a reduction in the bonding to bone in ceramic implants used to fill femoral defects in rabbits (RECK and BERNAL-SPREKELSEN, 1989). Another study found no significant increase in the blood supply nor in bone formation in a standardised defect of both tibiae filled with autologous graft material from the iliac crest in dogs (LuCH7 et al., 1986). Another study domonstrate a reduction in heterotopic osteoinduction in the abdominal musculature of rats (PINHOLTet al., 1992). At the current knowlegde, fibrin used alone in the fractures produces a retard allograft fusion mass formation (JARZEM et al., 1996); fibrin used as a carrier of inductive cytokines produces a higher yields of new bone (SATo et al., 1991); and fibrin used in a mixture containing apatite-wollastonite and glass ceramic granules (ONo et al., 1990), heterologous cancellous transplants (Bosh et al., 1980) or hydroxyapatite implants (BoNuccI et al., 1997) produced also a higher osteoinductive properties. However, the precise role that fibrin plays in these process is unclear. Thus, the purpose of this study was to evaluate the effects of the addition of SAF to HAP-coated titanium (Ti) implants on osteogenesis using radiographical and histo-morphometrical evaluations in a controlled rabbit model.

Materials and m e t h o d s Experiment design: Twenty-four New Zealand adult rabbits, 4~5 kg of weight, were randomly divided in four groups. The first group (Ti, control group) included 6 rabbits in which a titanium

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module was implanted. The second group (HAP, n = 6) received a titanium module a HAP coating. The third group (HAP+SAF, n = 6) received the HAP-coated Ti module, and a fibrin glue preparation was added. In the fourth group (SAF, n = 6), a Ti module was inserted, also with a fibrin glue preparation. Biomaterials and SAF: The implants used were two kinds of cylinder (DePuy BiolandSM), both measured 10 mm in length, and 3 mm in diameter. The first model was made of a Ti-6A1-4 V alloy and the second model of Ti-6A1-4 V with a HA coating whose calcium-phosphorous ratio (Ca-P) was 1.772 + 0.004. The fibrin preparation (Tissucol Inmuno ®) was made by two-component fibrin glue. The first component is a lyophilised protein concentrate containing fibrinogen, plasma fibronectin, factor XIII and human plasminogen, which is reconstituted with aprotinin solution; the second component is a preparation containing human thrombin, which is reconstituted with calcium chloride. Implantation procedure: The procedures followed in this study have been performed in accordance with national animal welfare legislation. All rabbits received anaesthesia induction using ketamine chlorhidrate (10 mg/kg IM, Imalgene 500, Rh6ne Poulanc), atropine sulphate (3 mg/kg IM), xylazine (5 mg/kg, Rompun, Bayer) and the surgical technique was performed under inhalation (isoflurane, 1.5-2%). Also, buprenorfine (0.015 mg/kg, Buprex, Shering Plough) was used as analgesia. An antibiotic prophylaxis (ampicillin, 10 mg/kg IM) was administred at the time of induction of anaesthesia. Then, the animals were weighed and subjected to preoperative radiographical examination of the hips. Posteriorly, each rabbit was placed on the operating table in the dorsal decubitus position and the hind limbs were desinfected with a chlorhexidine solution and both hips were isolated with sterile gauzes. An aperture of 3 cm approximately was made in the lateral aspect of the selected hip and the trochanteric area was exposed subperiostally. By means of a pneumatic motor, a perforation was made using a 3.2 mm drill, in the direction of the neck of the femur. Afterwards, an appropriate cylinder was introduced, using the material described in each group. In the group SAF, orifice made by the drill was filled, with Tissucol Inmuno 1.0 (Dulpoject®), which provided a seal for an area of at least 10 cm 2, after which the cylinder was introduced. A two layer wound closure was performed. No anti-inflammatory medication was administered afterwards. Movements of the animals was allowed after postoperatory period. Seven weeks later (the minimal period calculated for bone bonding) the animals were euthanised, in groups of six, using a lethal thiopental overdose and a radiographical study of the hips was performed (Fig. 1). Evaluation and treatment of the specimens: A subtrochanteric osteotomy was performed on each rabbit and afterwards, the specimen obtained was x-rayed (25 kW, 35 seconds) with a Cabinet X-Ray System unit, Faxitron ® Hewlett-Packard (USA), in order to determine the position of the cylinder. When it was identified, the bone was sectioned, using an Exakt ® diamond-dust coated saw.

When the cutting was complete, the procedure for embedding in methacrylate was initiated following the next phases: - Fixation: the samples were placed in formaldehyde solution for a week. - Gradual dehydration: the samples were exposed to ever-increasing concentrations of ethyl alcohol. - Infiltration: The samples were embebbed in a mixture of 100% ethyl alcohol and methacrylate (1:1) during one week. - Inclusion: Immersion in a plastic resin of pure polymethylmethacrylate Technovit 7200VLC ® during 3 weeks. - Polymerisation: change of the methacrylate from a liquid to solid state, by placing the samples in a Histolux ® Exakt light polymeriser, with 2 hours under white light and 4 hours under blue light.

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2. A-P x-ray of the specimen, showing the position of a Ti + HAP implant in the cervicotrochanteric region of the rabbit, six weeks after implantation. New bone formation in direct contact with the surface of the implant. Fig.

Fig. 1. Postoperative A-P X-Ray showing the position of the implant in the proximal femur.

When the methacrylate was polymerised, sections of 1 mm thick were obtained from the blocks, which were then polished to obtain a section of 12 ~tm approximately, and treated with a precision adhesive Technovit 7210 VLC ®. The sections were then stained using Goldner's trichrome technique and the yon Kossa stain, for quantitative microscopic evaluation of the static variables of bone remodelling according to the method described by PARFITTet al. (1987). The samples were studied with a LEICA ® stereo microscope (loupe), Model WILD M3Z (Switzerland), with a magnification of 16 × 10 = 160. In order to obtain the required measurements, using the linear interception and point counting techniques, a specific process was designed using image analysis equipment, composed by: - System unit: LEICA ®. Model Q500MC. Pentium, 1GB hard disc, 16MB memory, MS-Dos 6.22 system, Windows for Workgroups 3.11. Great Britain. - Imaging: Resolution (horizontal: 712 pixels) x (vertical: 512 pixels). Conversion scale 1 pixel = 4.05 square microns, with the 16 x 10 lens in the microscope. High resolution monitor, manufactured for LEICA ® by Liyama Electric Co. Ltd. ®. High resolution video camera mounted on the stereo microscope: DONPISHA ®, Model 3CCD colour Vision Camera Module, Japan. - Analysis and imaging tools: Software - QWin, Microsoft Dos and Windows for LEICA ®.

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The following parameters were studied in each section of the implant: Mean distance, defined as the distance between the formed new-bone or fibrous tissue and the implant (DM); Total bone volume (TBV), the total quantity of bone or osteoid tissue in each slide; Total volume of fibrous tissue (TFV), or the quantity of fibrous tissue in the field; Total volume of bone or fibrous in contact with the implant (IS); Total volume of bone tissue in contact with the implant (BS); Total volume of fibrous tissue in contact with the implant (FS); Percentage of bone in contact with the implant, obtained using the formula BS/IS xl00 (BS/IS [%]); Percentage of fibrous tissue in contact with the implant: FS/IS xl00 (FS/IS [%]). Radiological examination was also carried out to detect signs of bone bonding, defined as the presence of bone condensation or neoformation areas greater than 1 mm adjacent to the implant.

Statistical analysis In order to analyse the influence of the different treatments on each of the variables to be evaluated, the following model was used for covariance analysis: Yijk = 0 + [~ (weight)ijk + (HAP)i + (SAF)j + (HAP * SAF)ij + eijk ; id = 0.1

Where Yijkrepresents the value of each of the assessment variables evaluated on the kessimal experimental subject belonging to the i-essimal level of HAP and the j-essimal level of SAF, whose weight is O + [3(weight)ijk. (HAP) i and (SAF)j represent the main effects of the treatments, (HAP * SAF)ij represents the corresponding interactions, and eij k represents the random errors which we assume to be independent and evenly distributed N (0, or). In order to minimize the main effects, interactions and weight effect, the appropriate F-tests were used. A contrast was considered to be significant when P < 0.05. The R-square was calculated for all models. The models were estimated with the assessment variables evaluated in the original scale and logarithmically transformed. We chose the scale those gave the best adjustment based on the R-square. Finally, the adjusted means and measurements were obtained for each variable for each experimental group.

Results All the rabbits employed finished the study and no variation on weight was observed after surgery. There were no clinical signs of infection or other generalised complications. A diaphyseal fracture of the femur was observed radiographically in one rabbit, but it consolidated spontaneously and did not affect to the experience. Radiographical

findings

One animal belonging to the SAF group was presented with the implant protruding from the bone and was therefore excluded from the study. Radiographically, all the animals studied presented signs of trabecular bone neoformation surrounding the implant, but areas of greater density were most evident in the HAP group (Fig. 2). The formation of new extracortical bone was rare and not related to the different groups.

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Histo-morphometry of the bone/implant interface The mean and the adjusted mean were obtained for each of the remodelling parameters analysed in the different groups (Table 1). Histo-morphometric analysis showed signs of neoformation in the area around the implant in all animals. Direct contact between the new bone and the implant surface was observed in all groups, although in some cases it consisted of isolated lacunae of lamellar bone (Fig. 3). There was no statistical differences in the mean distance between the implant and the bone in the different groups, hence the H A P + S A F interaction did result in a closer contact (Table 2).

Table 1. Bone Remodeling Parameters around of the implants after six weeks of implantation. Mean and Adjusted Mean by variable between groups. Standart error and 95% confidence interval are given in parentheses. Dependent Variable TBV

TFV

IS

BS

FS

BS/IS (%)

FS/IS (%)

Ti N=5

HAP N=7

SAF N=3

HAP + SAF N=6

Mean a

15.34 (0.63)

15.57 (0.23)

15.61 (0.16)

15.18 (0.29)

Adjusteda Mean

15.34 (14.98-15.71)

15.57 (15.26-15.88)

15.61 (15.14-16.08)

15.18 (14.85-15.51)

Mean a

15.65 (0.18)

15.40 (0.33)

15.17 (0.69)

15.57 (0.25)

Adjusteda Mean

15.65 (15.33-15.96)

15.39 15.13-15.66)

15.19 (14.78-15.59)

15.57 (15.28-15.86)

Mean"

10.93 (0.13)

11.11 (0.22)

11.39 (0.41)

11.54 (0.13)

Adjusted~ Mean

10.93 (10.71-11.15)

11.11 (10.93-11.29)

11.39 (11.12-11.67)

11.54 (11.35-11.74)

Mean ~

8.24 (1.36)

9.57 (0.96)

9.43 (0.84)

9.37 (0.61)

Adjusteda Mean

8.24 (7.29-9.19)

9.57 (8.76-10.37)

9.44 (8.22-10.67)

9.37 (8.50-10.23)

Mean a

19456 (2796)

20809 (12521)

35623 (12352)

41611 (21173)

Adjusteda Mean

19443 (5654-33233)

20685 (9026-32344)

35871 (18056-53686)

41642 (29053-54230)

Mean"

1.91 (1.34)

3.06 (0.84)

2.6 (0.45)

2.42 (0.60)

Adjusteda Mean

1,91 (1.05-2.78)

3.06 (2.32-3.79)

2.65 (1.53-3.77)

2.42 (1.63-3.22)

Mean a

3.53 (0.24)

3.27 (0.66)

3.63 (0.25)

3.46 (0.87)

Adjusteda Mean

3.53 (2.93-4.14)

3.26 (2.75-3.77)

3.65 (2.86-4.43)

3.46 (2.91-4.02)

a Calculated according to the covariables in the model: Weight in kg: 2.81905

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M. MORALES et al.

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Effects of fibrin on the integration hydroxyapatite coating implants Table 2. Mean Distance between the bone and implant (DM). Groups

Mean

Std. Error

Ti HAP HAP + SAF SAF

4.256" 4.681 ° 4.456" 4.770"

0.251 0.213 0.229 0.325

109

Table 3. Statistical Significance by variable between groups. p-value

aCalculated according to the covariables in the model: Weight in kg: 2.81905

DM TBV TFV IS BS BS/IS(%) FS FS/IS(%)

HAP

SAF

HAP*SAF R-square

0.831 0.575 0.681 0.136 0.194 0.290 0.607 0.450

0.582 0.728 0.362 0.001 (**) 0.293 0.908 0.013 (*) 0.599

0.171 0.079 0.052 0.897 0.149 0.123 0.739 0.884

0.145 0.226 0.320 0.599 0.278 0.245 0.387 0.098

(*) p < 0.05; (**) p < 0.005 Seven weeks after implantation, the total area of formed new bone was similar in all groups (Ti 24.8%; H A P 25.2%; S A F 23.2%; H A P + S A F 24.6% (not significant) (Table 3). The presence of fibrous tissue in the peripheral areas of the implant was frequently seen, but in group SAF, the fibrous tissue formation was significantly greater (p = 0.001) and the gaps were greater (Fig. 4), compared with other groups (Fig. 5). There was no difference in the area of trabecular bone in direct contact with the implant in the different groups (TI, 22.5%; HAP, 26.1%; H A P + SAF, 25.6%; SAF, 25.7%). However, the area of fibrous tissue in contact with the implant was significantly greater in S A F (p = 0.013) (Fig. 6).

45000. / i 40000 35000

30000

o .E o. .~

2s000 2 0000 15000

t~ '~

10000 5000 0

,

Ti

,

HAP

#

HAP+SAF'

SAF

Fig. 6. Histomorphometric results of the Total Bone Volume (BS) and Total Fibrous Volume (FS). The formation of fibrous tissue in contact with the implant was significantly higher in Group SAF (fibrin alone).

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M. MORALESet al.

Discussion Based on our results, the model used in this study appears suitable for the evaluation of the osteogenic response induced seven weeks after implantation by the addition of SAF to HAP- coated Ti implants. The placement of the implants in the cervicotrochanteric region of the proximal femur, reflects a conditions very similar to those of biological fixation in the reconstruction of human joints. On the other hand, this study represents a controlled experimental situation in which the effects of the HAP + SAF combination are evaluated and they can also be compared with the effect of fibrin on each group and with the other control groups. Our results seem to indicate that HAP + SAF combination does not increase new-bone formation, with no differences encountered in the rate of bone growth compared to the control group. In addition, SAF gave rise to a significant increase in fibrous tissue compared to the other groups. A review of the literature available shows that the effect of fibrin on bone bonding with biomaterials remain unclear. Some authors have suggested that fibrin has stimulant effects on new bone growth. Thus, NAKAMURAet al. (1998) have reported that the injection of a mixture of HAP and fibrin into the rabbit femur produces a significant increase in new-bone formation compared with the fibrin-free group. Using histo-morphometric analysis, ONO et al. (1990) following implantation of ceramic crystals with apatite-wollastonite (A-W.GC) in defects created in the proximal tibia of rats, found a higher osteoconductive activity in the A-W.GC-Fibrin mixture than in A-W.GC without fibrin. KANIA et al. (1998) observed that the addition of fibrin to coral significantly increased the new bone formation in defects created in the femoral condyle of rabbits, compared to coral alone. The osteoinductive effect of fibrin has also been confirmed by other reports (KELLERet al., 1985; KAWAMURAand URIST,1988). Nevertheless, other authors have found no effects on increased osteogenesis, which are in agreement with our results. RECK and BERNAL-SPREKELSEN(1989), using semi-quantitative measurements, observed that the implantation in the rabbit femur of HAP granules with homologous fibrin, is associated with greater formation of soft tissue and with a reduction in the bonding to bone of ceramic implants. PINHOLTet al. (1992) reported that the implantation in the abdominal musculature of rats of a demineralised bone matrix combination and two substances, one a fibrin-collagen paste and the other a fibrin seal, produced chronic inflammation and an inhibition of osteoinduction. LOCHTet al. (1986)did not find a significant increase in new bone formation in a standardised defect in both tibiae of dogs that had been filled with a mixture of an autologous bone transplant from the iliac crest, and fibrin. Similar findings have been reported by other authors (LuNCHTet al., 1986; SHENet al., 1993; JARZEMet al., 1996). On the other hand, our results indicates the significant growth of fibrous tissue in contact with the implant in group SAF. Furthermore, the percentage of fibrous tissue in contact with the implant was also greater in group HAP + SAF. SATO et al. (1991) also observed that 8 weeks after injection of a compound of HAP fibrin and bone morphogenetic protein in defects of the femoral condyle in rabbits, there was a higher presence of

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fibrous tissue in the HAP and HAP + fibrin; they also found that in the SAF group, newbone formation was very poor and cavities were filled with adipose tissue and bone marrow. RECK and BERNAL-SPREKELSEN(1989) reported a higher degree of fibrous tissue formation following implantation of a mixture of HAP + fibrin in femoral defects in rabbits. Our study also showed that a greater fibrous tissue was found in the fibrin alone group (SAF). This fact can promote a decrease in the osteogenesis and, consequently, slows down the fracture repair. These controversial results obtained by the different authors could indicate that the fibrin influence on osteogenesis may cause a double effect. Under some circumstances, fibrin would be a part of the acute inflammatory reaction promoting the arrival of mesenchymal cells, precursors of bone, which would increase osteoblastic activity and favour osteogenesis (ScHWARZ, 1993). Under other circumstances, the fibrin would promote a foreign body inflammatory reaction, with activation of macrophages that, through the liberation of mediators, would stimulate fibroblast proliferation and increase fibrotic activity at the interface (RAGHOW, 1994; RECK and BERNAL-SPREKELSEN,1989). Further researchs are necessary to establish the influence induced of factors (biomaterials, nature of the coating, etc) or general factors (immune response), in order to preview the effects of the fibrin establishment in each case. In conclussion, the present study based on a rabbit model, seems to demonstrate that the addition of fibrin do not result in an increase in new-bone growth, as well as that it do not favour the closer contact observed using Ti implants, coated with HAP or uncoated. Furthermore, the addition of fibrin was accompanied with a higher degree of fibrous tissue formation.

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KAN1A,R.E., A. MEUNIER,M. HAMADOUCHE,L. SEDEL,and H. PETITE. 1998. Addition of fibrin sealant to ceramic promotes bone repair: long-term study in rabbit femoral defect model. J. Biomed. Mater Res. 43 (1): 38-45. KAWAMURA,M., and M.R. URIST. 1988. Human fibrin is a physiologic delivery system for bone morphogenetic protein. Clin. Orthop. 235: 301-310. KEAVENY,T.M., and D.L. BARTEL.1995. Mechanical consequences of bone ingrowth in a hip prosthesis inserted without cement. J.. Bone. Joint. Surg. Am. 77 (6): 911-923. KELLER,J., T.T. ANDREASSEN,F. JOICE,V.E. KNUDSEN,P.H. JORGENSEN,and U. LUCHT. 1985. Fixation of osteochondral fractures. Fibrin sealant tested in dogs. Acta Orthop. Scan& 56 (4): 323-326. LUC~T, U., C. BUNGER,J.T, MOLLER,F. JOYCE, and H. PLENKJr. 1986. Fibrin sealant in bone transplantion. No effects on blood flow and bone formation in dogs. Acta Orthop. Scand. 57 (1): 19-24. NAKAMVRA, K., T. KOSHINO, and T. SAITO. 1998. Osteogenic response of the rabbit femur to hydroxyapatite thermal descomposition product-fibrin glue mixtura. Biomaterials 19 (20): 1901-1907. ONO, K., T. YAMAMURO,T. NAKAMURA,and T. KOKUBO. 1990. Apatite-wollastonite containing glass ceramic granule-fibrin mixture as a bone graft filler: use with low granular density. J. Biomed. Mater Res. 24 (1): 11-20. PARFITT, A.M., M.K. DREZNER, F.H. GLORIEUX,J.A. KANIS, H. MALLUCHE,P.J. MEUNIER, S.M. OTT, and R.R. RECKER. 1987. Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASMBR Hismorphometry Nomenclature Committee. J. Bone Miner. Res. 2: 595-610. PIDHORZ,L.E., R.M. URBAN,J.J. JACOBS,D.R. SUMMER,and J.O. GALANTE.1993. A quantitative study of bone and soft tissues in cemenfless porous-coated acetabular components retrieved at autopsy. J. Arthroplasty 8: 213-225. PINHOLT,E.M., E. SOLHEIM,G. BANG,and E. SUDMANN.Bone induction by composites of desmineralized bone and rats: a comparative study of fibrin-collagen paste, firin sealant, and polyorthoester with gentamicin. J. Oral Maxillofac. Surg. 50 (12): 1300-1304. RAGHOW,R. 1994. The role of extracellular matrix in postinflammatory wound healing and fibrosis. FASEB J. 8: 823-831. RECK, R., and M. BERNAL-SPREKELSEN.1989. The effect of fibrin glue on the healing of hidroxyapatite ceramics. An animal experiment study. HNO 37 (3): 112-116. SATO, T., M. KAWAMURA,K. SATO,H. IWATA,and T. MIURA. 1991. Bone morphogenesis of rabbit bone morphogenetic protein-bound hydroxyapatite-fibrin composite. Clin. Orthop. 263: 254-62. SCHWARZ,N. 1993. The role of fibrin sealant in osteoinduction. Ann. Chir. Gynaecol. SuppL 207: 63-68. SCHWARZ,N., H. REDL,L. ZENG,G. SCHLAG,H.P. DINGES,and J. ESCHBERGER.1993. Early osteoinduction in rats is not altered by fibrin sealant. Clin. Orthop. 293: 353-359. SHEN, W.J., K.C. CHUNG,G.L. WANG, G. BALIAN,and R.E. McLAUGHLIN. 1993. Demineralized bone matrix in the stabilization of porous-coated implants in bone defects in rabbits. Clin. Orthop. 293: 346-352. SOBALLE, K., E.S. HANSEN, H. BROCKSTEDT-RASMUSSEN,and C. BUNGER. 1990. Hydroxyapatite coating enhances fixation of porous coated implants: A comparison in dogs between press-fit and noninterference fit. Acta Orthop. Scand. 61: 299-306. SOBALLE, K., E.S. HANSEN,H. BROCKSTEDT-RASMUSSEN,C.M. PEDERSEN, and C. BUNGER. 1993. Hydroxyapatite coating converts fibrous tissue to bone around loaded implants. J. Bone Joint Surg. 75 (B) : 270-278.

Correspondence author: CARLOS GUTIERREZ,Veterinary Faculty, University of Las Palmas de Gran Canaria, 35416, Arucas, Las Palmas, Canary Islands, Spain Tel: ++34 928451115, Fax: ++34 928451142; e-mail: [email protected]