A histomorphological study on self-reinforced polyglycolide (SR-PGA) osteosynthesis implants
coated with slowly absorbable polymers Jarkko Vasenius,* Seppo Vainionpaa, Kimmo Vihtonen, Matti Mero: Antero Makela, Pertti Tormala) and Pentti Rokkanen Department of Orthopaedics and Traumatology, Tiiolo Hospital, Helsinki University Central Hospital, Topeliuksenkatu 5 SF-00260 Helsinki; 'The College of Veterinary Medicine, Hameentie 57, SF-00550 Helsinki; fBiomaterials Laboratory, Institute of Plastics Technology, Tampere University of Technology, PO. Box 527, SF-33101 Tampere, Finland A total of 150 osteotomies of rabbits' distal femur were fixed with absorbable selfreinforced polyglycolide (SR-PGA) rods coated with slowly absorbable polymers. In order to reduce the degradation rate of the SR-PGA construction rods were coated with n-butyl-2-cyanoacrylate, polydioxanone (PDS), poly-beta-hydroxybutyrate (PHBA), or poly-1-lactide (PLLA).Biocompatibility and the rate of biodegradation were evaluated in histological, histomorphometric, microradiographic, and oxytetracycline labeling studies. PDS, PHBA, and PLLA coatings showed good biocompatibility although there were scattered fluid accumulations around the
rod in each group. Cyanoacrylate coating seemed to inhibit cartilage regeneration and cause the formation of considerable amount of connective tissue around the implant. The PGA core of the rod had totally degraded in 24 to 36 weeks in each coating group. Cyanoacrylate and PDS coatings were not detectable after 6 weeks while PHBA and PLLA coatings were still observed after 48 weeks. There were five (14%) non-unions in the cyanoacrylate coating group, one (3%)in the PHBA coating group caused by a purulent infection and none in the PDS and PLLA coating groups.
INTRODUCTION
Absorbable polyglycolide (PGA) osteosynthesis implants were introduced by Schmitt and Polistina 1969.' PGA is widely known as the first synthetic absorbable suture material (Dexon).' Its biocompatibility and biodegradation in soft and hard tissue has been proven in several experimental and clinical Rokkanen et al. presented the first clinical series in which absorbable PGA/ PLA rods and sutures were compared with conventional metallic screws and plates in the fixation of malleolar fractures.' In both groups the results were equally favorable, but unlike the absorbable implants metallic implants had to be removed operatively after the fracture had healed. During developing ab*To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 24, 1615-1635 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/121615-21$04.00
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sorbable osteosynthesis implants about 2000 animals have been operated in experimental trials and more than 900 patients have been treated with absorbable osteosynthesis implants at our department so far. The excessive rigidity of the fracture fixation performed with the metallic implants causes osteoporosis and atrophy of the cortical bone underneath the implant?" This may lead to secondary fractures after the removal of 0steosynthesis implants especially from diaphyseal bone. The elasticity modulus of PGA implants (10-15 GPa)'' is close to that of cortical bone (6-20 GPa)"12 which diminishes the risk of such disadvantages. The initial bending and shear strength of SR-PGA rods (200 5 25 MPa and 350 k 50 MPa)13,'4have been superior to other absorbable devices presented in the 1iterat~re.l~'~ However, PGA is the most hydrophilic absorbable polymer and SR-PGA rods lose their mechanical properties in 6-8 weeks in 37°C distilled water and in 5-6 weeks in the subcutis of rabbits.I4For this reason the clinical use of SR-PGA osteosynthesis implants has been limited to the fixation of fractures and osteotomies which heal relatively fast and where the fixation site is not exposed to hard stress during fracture healir~g.~,'~ Hydrolysis in aqueous environment is considered the main pathway of degradation of PDS, PGA, PHBA, and PLA, but the initiation of this process could be enhanced by enzymatic activity at least with PGA and PLA.'9-21The end products of PDS, PGA, PHBA, and PLA degradation are excreted in exhaled carbon dioxide (COJ and in urine.2'22-25 N-butyl-2-cyanoacrylate is degraded into formaldehyde and alkylcyanoacetate.26Polydioxanone (PDS), poly-beta-hydroxybutyrate(PHBA) and polylactide (PLA) have shown good biocompatibility in soft and hard t i s ~ u e . ~ ~They ~ ' - ~all " degrade slower than PGA .23,31-33 N-butyl-2-cyanoacrylate is better known as an absorbable tissue adhesive (Histoacryl).Unlike its monomer form n-butyl-2-cyanoacrylate polymer has been well tolerated by bone The aim of the present study was to evaluate the biocompatibility and absorption rate of the SR-PGA rod coated with four different slowly absorbable polymers after implantation in cancellous bone of rabbit. Rods were coated in order to decrease their degradation rate. EXPERIMENTAL METHODS
Implant materials Cylindrical self-reinforced polyglycolide (SR-PGA) implants were manufactured by partially melting and sintering commercial PGA sutures (Dexon " S size USP 2, Davis Geck, Gosport, U.K.) at temperatures 205"C-232"C at elevated pressure. In the coating process SR-PGA rods were five times briefly immersed in 55°C solutions of chloroform containing 0.75% and 1.5% PDS (Ethicon, Norderstedt, FRG), PHBA (Marlborough Biopolymers, L td., Cleveland, U.K.) or poly-1-lactide (PLLA; CCA Biochem b.v. Gorinchem, The Netherlands). After the immersion the chloroform was evaporated leaving a
+
SR-PGA IMPLANTS COATED WITH POLYMERS
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30-40-pm thick continuous polymer layer on the rod. A cyanoacrylate coating of equal thickness was laid down at room temperature by immersing the rod briefly in n-butyl-2-cyanoacrylate monomer (Histoacryl blau, B. Braun Melsungen, West Germany) which polymerized on the rod. The thickness of the coating was evaluated by measuring the diameter of the rod before and after the coating process. The nominal diameter of the coated rod was 3.4 mm. Animal experiments and preparation of specimen A total of 150 adult rabbits of both sexes weighing 2400-4400 g were operated on. Rabbits received 0.5 mg/kg atropine (Atropin) subcutaneously as premedication and they were anesthetized with subcutaneous medetomidine (Domitor) 0.3 mg/kg, ketamine (Ketalar) 0.4 mg/kg, and diazepam (Diapam) 0.2 mg/kg. They were also injected subcutaneously 150000 IU procaine penicillin (Procapen) as infection prophylaxis. The right hind leg was shaved and scrubbed with antimicrobial solvent (Neo-Amisept).An anteromedial incision was made and patella was dislocated laterally. A transversal osteotomy was made in the cancellous bone of distal femur with a circular saw. A longitudinal drill channel, 3.4 mm in diameter and 2.5 mm long, was made in the intercondylar area. The osteotomy was fixed with coated PGA implant applied in the drill channel. The fixation was secured with 2-3 USP size 0 PGA sutures (Dexon, Davis + Geck, Puerto Rico) running through transversal drill channels made on the both sides of the osteotomy (Fig. 1 A,B). Openings were closed with PGA sutures and rabbits were returned to their cages and they received regular laboratory animal diet and water. No external support was used. The follow-ups and the number of rabbits followed is presented in Table I. Rabbits were sacrificed with an overdose of f luanizone (Hypnorm) and an intravenous air embolism. Three days before sacrifice they were injected with 50 mg/kg oxytetracycline (OTC; Terramycin) for fluorescence After sacrifice both femurs were exarticulated, dissected, and radiographs were taken in anteroposterior and lateral views. Any signs of infection or other complication were visually controlled. The distal portions of both femurs were fixed in a series of ethanol immersions with rising concentrations (70-99%) and then embedded in methylmetha~rylate.~~ The intact femurs’ served as control specimens. Longitudinal sections, 5 pm thick, were cut in frontal plane with microtome (Jung, Polycut S, 1983) and stained by using Mason-Goldner’s method for histological and histomorphometrical evalua t i ~ nFor . ~ OTC-f ~ luorescence and microradiographic studies longitudinal sections in frontal plane, 80 p,m thick, were prepared with saw microtome (Leitz 1600). In the evaluation of OTC-fluorescence the intensity of fluorescence was graded from 0 to 3 and the fluorescence index was calculated by subtracting the intensity of the intact control specimen from the operated one. In microradiography Kodak Professional film SO 343 (Eastman Kodak, Rochester, NY) and 50 kV, 9 mA, 12 min exposure time and 29.5 cm film-focus were used. In quantitative histomorphometric analysis, a light microscope, (Leitz Wezlar,
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.*imp1 ant
.. s u t u r e
(b) Figure 1. (a) Schematic frontal view of distal femur showing horizontal osteotomy, implant, and the suture pulled through drill channels which were drilled at the proximal and distal aspects of the osteotomy. (b) Side view of distal femur showing osteotomy, drill channels, the implant, and the suture.
Ernst Leitz Co., Wetzlag, FRG) linked via a video camera to a semiautomatic computerized analysis system, MOP-Videoplan (Kontron, Munich, FRG) was used. A magnification of x16 was used and the magnification of the computer itself was ~ 2 5 0 The . microscopic field was displayed on TV screen of the computer and measurements were performed by using a digitizer tablet and cursor linked to it. The area of cartilage at the entry of the drill channel was measured to analyze cartilage regeneration. The areas of connective tissue, trabecular bone and new bone around the implant, as well as the implant area were measured from three consecutive fields along the implant (Fig. 2). TABLE I Plan of Experimental Studies on Rabbits Number of rabbits followed up (weeks) Implanted SR-PGA rod
1
3
6
12
24
36
48
C yanoacrylate-coated PDS-coated PHRA-coated PLLA-coated
5 5 5 5
5 5 5 5
5 5 5 5
5 5 5 5
5 7 5 5
5 8 5 5
8 5 6 6
38 40 36
20
20
20
20
22
23
25
150
Total ~
Totalnumber of rabbits
36
SR-PGA IMPLANTS COATED WITH POLYMERS
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Measured parameters A:
cartilage connective tissue implant new bone trabecular / subchondral bone
B-C: connective tissue implant new bone trabecular bone
D:
callus bone connective tissue trabecular bone
Figure 2. Schematic frontal view of the right femur. The histomorphometric measurements were performed in four standardized areas (A-D). The left femur served as control and was studied in the same manner.
The area of callus bone, connective tissue, and trabecular bone in the medial condyle at the level of osteotomy were measured to analyze the consolidation of the osteotomy. In statistical analysis regression analysis and x2test were used. P value less than 0.05 was considered statistically significant. RESULTS
Macroscopic studies The implants were radiolucent and drill channels for the rod were difficult to detect. However, channels drilled for the sutures were still detectable 48 weeks after operation. Radiographs showed a bony union of osteotomies in 6 weeks, but the osteotomy was partly detectable up to 24 weeks in each group. A Failure of fixation with a displacement of fragments was found in five (14%)rabbits in the cyanoacrylate coating group (24 and 48 weeks after operation) and in one rabbit in the PHBA coating group (6 weeks after operation). The latter was due to a purulent arthritis. A purulent arthritis was detected in two other operated knee joints but the osteotomies had consolidated normally in those cases. Six rabbits had to be sacrificed prior the end point of the follow-up period due to postoperative respiratory infection or trauma. Histological and microradiographic studies
One week Two out of five osteotomies treated with cyanoacrylate or PHBA coated rods and three out of five osteotomies treated with PDS or PLLA coated rods showed a bony union in histological and microradiographic studies. Most of
VASENIUS ET ALL.
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the coatings had detached from the rod and strips of coating material were found at the entrance of the drill channel (Fig. 3). Early signs of biodegradation of PGA core were found only in the cyanoacrylate coating group. PLLA coating had retained the best on the rod. A strong new bone formation was found in the metaphysis, in the periosteum, and around the osteotomy. A thin layer of granulation tissue covered the entrance of the drill channel in each group.
Three weeks All osteotomies, except one that was treated with PHBA coated rod, showed a trabecular bone union in histological and microradiographic studies (Fig. 4).A strong new bone formation was found around the osteotomy, in the periosteum and in the metaphysis. A thin layer of new bone had initiated to grow on the rod in each group. More granulation tissue, some cartilage, and new bone had grown at the entrance of the drill channel. Early signs of degradation of the PGA core were detected at the periphery of the rod especially where the coating had been detached. Most of the coatings had detached from the rod, but one or two samples of each rod type had an entire coating. Scattered small fluid accumulations with phagocytizing macrophages were found attached to the rod in each group.
Figure 3. Curly strips of PLLA coating (*) detached from the PGA implant (I) at the entry of the drill channel one week after operation. (MassonGoldner, original magnification ~ 1 0 0 . )
SR-PGA IMPLANTS COATED WITH POLYMERS
(e)
Figure 4. A bony union of the osteotomy partly through external callus formation and partly through trabecular bone formation was found three weeks after operation. Note the formation of new bone around the implant (I). (Masson-Goldner, original magnification X5.)
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Six weeks All osteotomies except one had healed and were difficult to locate histologically and microradiographically. One fixation with a PHBA coated rod had failed due to an infection. Cyanoacrylate and PDS coatings were no longer found on rods. PHBA and PLLA coatings were observed, but they were mostly detached from the rod. The more the rod had coating attached the less it had degraded (Fig. 5).Macrophages with PGA in their cytoplasm were found inside and around the degrading implants in two samples in the cyanoacrylate coating group. In each group most of the rods had a lining of new bone on them. A thin layer of osteoid and connective tissue had formed between the rod and new bone. No signs of delayed inflammation reaction or foreign body reaction were observed. 12 weeks
All osteotomies had healed partly through trabecular bone union and partly through external callus formation. C yanoacrylate coated rods had degraded into a homogenous, liquid PGA mass. Reinforcing PGA fibers with granulation tissue intruding between them were observed in the other groups (Fig. 6). PGA core had degraded faster in the subchondral area than in the proximal metaphysis, Two PDS coated rods showed small expansions toward bone tissue or medullary cavity, which could be an early sign of sinus forma-
Figure 5. An undamaged PLLA coating (+) and new bone (B) on the PGA implant (I) showing only early signs of degradation 6 weeks after operation. (Masson-Goldner, original magnification ~ 2 0 . )
(e)
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Figure 6. Granulation tissue intruding between degrading PGA fibers (I) 12 weeks after operation. (Masson-Goldner, original magnification x40.)
tion (Fig. 7). Several macrophages were found in these fluid accumulations indicating only a nonspecific inflammation and phagocytosis of polyglycolide debris. New bone formation was found in the drill channel at the level of the osteotomy and at the entrance of the drill channel, that was still filled with connective tissue and some cartilage. At this follow-up the amount of bone and connective tissue around the implant was the highest. 24 weeks
Two osteotomies treated with cyanoacrylate coated rods showed a pseudoarthrosis, while all the other osteotomies had healed uneventfully. There were only remnants of PGA core found at the implant site, which was filled with granulation and connective tissue. There were two exceptions in the PHBA and PLLA coated rods with solid PGA fibers left and an almost entire coating around the implant. PHBA and PLLA coatings were still found at the implant site and bone tissue had grown on both sides of PHBA coating. The implant site was covered with bone or connective tissue. Cartilage and connective tissue filled still the narrow entrance of the drill channel. No signs of foreign body reaction or prolonged inflammation were observed. 36 weeks All osteotomies had healed uneventfully. PHBA and PLLA coatings were still present at the implant site. One rod with an undamaged PHBA coating
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Figure 7. An exceptionally fast degraded PDS coated SR-PGA implant and a fluid accumulation with a connective tissue lining (+) expanding into the bone marrow was detected 12 weeks after operation. Note the considerably faster degradation of the implant in the metaphysis than in the marrow cavity. (Masson-Goldner, original magnification x5.)
SR-PGA IMPLANTS COATED WITH POLYMERS
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had degraded exceptionally slowly while the rest of the rods were practically totally degraded. Bone and connective tissue lining around the implant site had been absorbed and shrunk toward the metaphysis (Fig. 8). There was still a gap in the bone structure at the entry of the drill channel which was filled with connective tissue and cartilage. One sample in the PDS coating group showed a foreign body reaction with dense focuses of lymphocytes. 48 weeks Three samples in the cyanoacrylate coating group showed a pseudoarthrosis with a displacement of one or both condyles. All the other osteotomies had healed uneventfully. PGA had been totally absorbed in each sample. Strips of PHBA and PLLA coating were still observed among bone and connective tissue (Fig. 9). The entrance of the drill channel was filled with bone iq a third of samples and with cartilage and connective tissue in the others. The trabecular bone tissue around the implant site resembled intact bone. Bane layer and connective tissue around the implant had been absorbed or immigrated toward the metaphysis in all groups except in the cyanoacrylate coating group where the amount of connective tissue around the implant remained high. The regeneration of cartilage had advanced well in all groups except in the cyanoacrylate coating group. Bone remodeling was still in progress.
Figure 8. A considerable amount of connective tissue (C) and regular bone marrow replaced the cyanoacrylate coated SR-PGA rod 36 weeks after operation. (Masson-Goldner, original magnification x 5.)
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VASENIUS ET AL
Figure 9. 48 weeks after operation remodeling was almost finished. Dense connective tissue and regular trabecular bone was found at the distal implant site and at the entry of the drill channel Note the strips of PLLA coating (+) in the connective tissue. (Masson-Goldner, original magnification ~5 and ~ 2 0 . )
(e).
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SR-PGA IMPLANTS COATED WITH POLYMERS
Histomorphometrical studies In each coating group the rod area had increased about 10% between the first and the third week (Fig. 1OA). In the PDS and PLLA coating groups the rod area started to decrease after 3 weeks and after 6 weeks in the cyanoacrylate and PHBA coating groups. At 12 and 24 weeks the area of PHBA coated rods was significantly larger compared to the rest of the rods ( p < 0.01). At 36 weeks the area of all implants, except one PHBA coated rod that was deleted from calculations, had decreased to zero. The area of connective tissue around the implant was largest between 12th and 36th weeks post implantation (Fig. 10B). In the cyanoacrylate coating group the amount of connective tissue remained high while it decreased close to zero in all the rest of the groups. The amount of cartilage at the entry of the drill channel returned close to that of the intact control side in all groups except in the cyanoacrylate coating group (Fig. 1OC). The amount of new bone in the medial callus reached the highest value three to 6 weeks after operation and de-
Implant area (rnm2, 30 Qanoacryiate coated 25
T
i
L
PDS coated PHBA coated
20
PLLA coated 15
10 1 5
0
I
I
1
3
6
12
24
36
48
Implantationtime (weeks) (a)
Figure 10. The histomorphometric analysis showed that the area of all rod types increased during the first 3 to 6 weeks and decreased to zero in 24 to 36 weeks (a). The amount of connective tissue around and in the implant reached its top between 6th and 12th weeks after operation and then started to decrease in all groups except in the cyanoacrylate coating group where the largest amount of connective tissue was found 36 and 48 weeks after operation (b). The amount of cartilage tissue at the entry of the drill channel was significantly smaller in the cyanoacrylate coating group than in the other groups (c). (* = cyanoacrylate vs. other coatings, p < 0.01. ** = PHBA vs. other coatings, p c 0.01).
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Connective tissue area (rnd) 25
20
15
10
5
0 1
3
6
12
24
Implantation time (weeks) (b)
2 Cartilage area (mm ) 4
3
2
1
0
Figure 10. (continued)
creased with remodeling of the bone. The amount of trabecular bone around the implant and did not show any significant differences between follow-up periods or coatings.
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SR-PGA IMPLANTS COATED WITH POLYMERS
Oxytetracycline fluorescence labeling studies The results of OTC fluorescence labeling studies are presented in Fig. 11 AD. Fluorescence in the operated specimen showed the highest intensity and thus strong new bone formation during the first 6 weeks in the periosteum, at the osteotomy, and in the trabecular bone, while at the entry of the drill channel for the implant it was lower than that of the intact specimen during the first 3 weeks increasing toward the 12th and 24th week, indicating rather late bone remodeling at that area. DISCUSSION
Diverging results of the degradation rate of PGA in bone tissue have been presented. Cutright et al. presented that only 17% of the PGA implanted in bone tissue had degraded in 31 weeks. C-14 labeling studies showed that 2.7% of the fast-cured PGA implant remained after 5 months, while 29.5% of the slow-cured PGA implant was left after 9 month^.^ Vainionpaa presented that only remnants of injection molded PGA implant were detected 12 weeks after implantation in cancellous bone.' In the present study the cyanoacrylate, PDS, and PLLA coated SR-PGA implants had degraded in 24 to
Periosteum Fluorescence 3,5
I
I Cyanoacrylate coated
31-
PDScoated
0 PHBAcoated PLLAcoated
?
3
6
12
24
36
48
Follow-up (weeks) (a)
Figure 11. Results of the OTC fluorescence studies. The intensity of OTC fluorescence was graded from 0 to 3 and the fluorescence index was calculated by subtracting the intensity of the intact control specimen from the operated one (0 = no fluorescence, 1 = weak fluorescence, 2 = moderate fluorescence, 3 = strong fluorescence).
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VASENIUS ET AL.
Osteotomy Fluorescence Cyanoacrylatecoated PDScoated
c]PHBAcoated
1
3
6
12
24
36
48
Follow-up (weeks) (b)
Subchondral bone Fluorescence 2
1
1
3
6
12
24
cyanoacrylate coated
36
b
48
Follow-up (weeks) (4 Figure 11. (continued)
36 weeks and PHBA coated implants in 36 weeks with one exception. At 12 to 24 weeks PHBA coated rods showed significantly slower degradation, which may be due to slightly better but still insufficient adhesion between PHBA coating and the SR-PGA core.
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Entry of the drill channel Fluorescence
Follow-up (weeks} (4 Figure 11. (continued)
Compared to the other three coating groups a higher failure rate of fixation of the osteotomy, a larger amount of connective tissue around the implant, and the lesser amount of cartilage at the entry of the drill channel were observed in the cyanoacrylate coating group. These adverse effects of cyanoacrylate coating may be due to its degradation product formaldehyde which is a toxic material and they make doubtful the biocompatibility of cyanoacrylate coating. In the present study strips of PHBA and PLLA coating were detected at the implant site still 48 weeks after implantation, which confirms the very slow degradation of PHBA and I'LLA.3233,38Some authors doubt about the biodegradation of PHBA.39 An identical fixation method with the present study was used by Vainionpaa et a1.40Implants used in their study were injection molded PGA rods without fiber reinforcement or coating. In their series all osteotomies healed uneventfully and the implants were replaced by bone tissue in 12 weeks. Vihtonene et al. showed that 71% of distal femoral osteotomiesfixed with pure PGA threads healed ~neventfully.~'Fixation of such osteotomies with pure I'GA rod has not been studied. In the present study 86% of the osteotomies fixed with cyanoacrylate coated rods showed undisturbed healing, while in the PDS and PLLA coating groups all osteotomies healed normally. In the PHBA group there was one non-union caused by a purulent infection, while the rest of the samples showed an uneventful healing. In the present study the area and thus the volume of the implant enlarged during the first 3 to 6 weeks. Scattered fluid accumulations and some expansions of degrading PGA toward the bone tissue were observed around the
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VASENIUS E’T AL.
implant in each group. These may be early signs of transient sinus formation, a phenomenon which has been reported in clinical studies on PGA and PGAI PLA sutures and PGA PGA is a very hydrophilic polymer and it absorbs fluids and expands as it degrades. This causes pressure on the surrounding tissues and a sinus may develop to the weakest point of the surrounding tissue. When the fixation of a fracture is concerned this would be the soft tissue around the mouth of the drill channel and the soft tissue around it. A transient sinus formation has never been reported in experimental studies on PGA rods. Unlike most clinical series the entry of the drill channel for the PGA rod has located intra-articularly in experimental studies. The joint capsule can resist hydrostatic pressure much better than scar tissue. Also the synovial tissue can absorb intra-articular fluid very well thus lowering the hydrostatic pressure in the capsule. According to the present study the degradation of coated SR-PGA rods takes 24-36 weeks which is 12-24 weeks longer than it takes injection molded PGA implants to degrade in cancellous bone tissue.6This difference seems to be mainly due to the self-reinforced structure and not due to the coating, because the adhesion between the PGA implant and any of the coatings used in the present study was insufficient for the implantation of coated rods in bone tissue. The majority of coatings had cracked and peeled at the implantation. Ethylene oxide sterilization may further weaken this adhesion. In our earlier study slowly absorbable coatings were able to improve statistically the strength retention of SR-PGA rods in the in vitro but not in the in vivo environment^.'^ The results of the present and our earlier studies have shown that slowly absorbable coatings which have a chemical structure which differs from that of the self-reinforced core can not provide major improvements to absorbable osteosynthesis implants. This study was supported by grants from the Foundation for Orthopaedic and Traumatology in Finland, from the Paulo Foundation, and from the Academy of Finland. We thank the following persons for technical assistance: Ms. H. Kuisma, Mr. J. Laiho, Mr. M. Tamminmaki, and Mr. J. Mikkola. The Finnish national guidelines for the care and use of laboratory animals has been observed in the present study.
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SR-PGA IMPLANTS COATED WITH POLYMERS 5. 6.
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D. E. Cutright, B. Perez, J. D. Beasley, W. J. Larson, and W.R. Posey, ”Degradation rates of polymers and copolymers of polylactic and polyglycolic acids,” Oral Surg., 37, 142-152 (1974). S. Vainionpaa, “Biodegradation of polyglycolic acid in bone tissue: An experimental study on rabbits,” Arch. Orthop. Trauma Surg., 104, 333338 (1986). 0. Bostman, S. Vainionpaa, E. Hirvensalo, A. Makela, K. Vihtonen, P. Tormala, and P. Rokkanen, “Biodegradable internal fixation for malleolar fractures,” J. Bone Jt. Surg., 69-B, 615-619 (1987). P. Rokkanen, P.O. Bostman, S. Vainionpaa, K. Vihtonen, P. Tormala, J. Laiho, J. Kilpikari, and M. Tamminmaki, ”Biodegradable implants in fracture fixation: Early results of treatment of fractures of the ankle,” Lancet, 1, 1422-1424 (1985). A. J. Tonino, C. L. Davidson, P. J. Klopper, and L.A. Linclau, “Protection from stress in bone and its effects. Experiments with stainless steel and plastic plates in dogs,” J. Bane Jt. Surg., 58-B, 107-113 (1976). P. Paavolainen, P. Slatis, E. Karaharju, and T. Holmstrom, ”Studies on mechanical strength of bone. 11, Torsional strength of cortical bone after rigid plate fixation with and without compression,” Acta Orthop. Scund., 49, 506-511 (1978). P. Tormala, P. Rokkanen, J. Laiho, M. Tamminmaki, and S. Vainionpaa, ”Material for osteosynthesis devices,” US.Pat. No. 4 743 257, 1988. B.S. Mather, “The symmetry of the mechanical properties of the human femur,” J. Surg. Res., 7, 222-225 (1967). S. Vainionpaa, J. Kilpikari, J. Laiho, P. Helevirta, P. Rokkanen, and P. Tormala, “Strength and strength retention in vitro, of absorbable, self-reinforced polyglycolide (PGA) rod for fracture fixation,” Biornaterials, 8, 46-48 (1987). J. Vasenius, S. Vainionpaa, K. Vihtonen, M. Mero, J. Mikkola, I? Rokkanen, and P. Tormala, ”Biodegradable self-reinforced polyglycolide (SR-PGA) composite rods coated with slowly biodegradable polymers for fracture fixation. Strength and strength retention in vitro and in vivo,” Clin. Muter., 4, 307-317 (1989). K. L. Gerlach and J. Eitenmiiller, “In vivo evaluation of eight different polymers for use as osteosynthesis material in maxillo-facial surgery,” in Biomaterials and Clinical Applications, A. Pizzoferrato, P. G. Marchetti, A. Ravaglioli, and A. J.C. Lee (eds.), Elsevier Science Publishers B.V, Amsterdam 1987, pp. 439-445. J.W. Leenslag, A. J. Pennings, R. R. Bos, E R. Rozema, and G. Boering, ”Resorbable materials of poly (L-lactide). VII. In vivo and in vitro degradation,” Bimaterials, 8, 311-314 (1987). P. Tormala, S. Vainionpaa, J. Kilpikari, and P. Rokkanen, “The effects of fiber reinforcement and gold plating on the flexural and tensile strength of PGA/PLA copolymer materials in vitro,” Bimaterials, 8, 42-45 (1987). P. Axelson, J. Raiha, K. Sittnikow, K. Skutsnabb, M. Mero, S. Vainionpaa, P. Tormala, and P. Rokkanen, “The use of a biodegradable implants in the fixation of small animal cancellous bone fractures,” Acta Vet. Scand., 29, 469-476 (1988). D. F. Williams and E. Mort, ”Enzyme accelerated hydrolysis of polyglycolic acid,” 1. Bioengin., 1, 231-238 (1977). D. F. Williams, ”Enzymic hydrolysis of polylactic acid,” Eng. Med., 10, 5-7 (1981). A.M. Reed and D. K. Gilding, ”Biodegradable polymers for use in surgery- poly( glycolic acid)/poly(lactic acid) homo and copolymers: 2. In vitro degradation,” Polymer, 22, 494-498 (1981).
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22. J. M. Brady, D. E. Cutright, R. A. Miller, and G.C. Battistone, “Resorption rate, route of elimination, and ultrastructure of the implant site of polylactic acid in the abdominal wall of the rat,” J. Biomed. Mater. Res., 7, 155-166 (1973). 23. J.A. Ray, N. Doddi, D. Regula, J. A. Williams, and A. Melveger, “Polydioxanone (PDS), a novel monofilament synthetic absorbable suture,” Surg. Gynecol. Obstr., 153, 497-507 (1981). 24. J.O. Hollinger, “Preliminary report on the osteogenic potential of a biodegradable copolymer of polylactide (PLA) and polyglycolide (PGA),” J. Biomed. Matev. Res., 17, 71-82 (1983). 25. A. L. Lehninger, in Principals of Biochemistry, S. Anderson, J. Fox (eds.), Worth Publishers, New York, 1982,pp. 524-526. 26. F. Leonard, R.K . Kulkarni, G. Brandes, J. Nelson, and J. J. Cameron, “Synthesis and degradation of poly (alkyl alpha-cyanoacrylates),” 1. Appl. Polym. Sci., 10, 259-272 (1966). 27. J. N. Baptist, ”Absorbableprosthetic devices and surgical sutures,” G.B. Pat. No. 1 034 123,1966. 28. R.K. Kulkarni, K.C. Pani, C. Neuman, and F. Leonard, “Polylactic acid for surgical implants,” Arch. Surg., 93, 839-843 (1966). 29. D. E. Cutright, E. E. Hunsuck, and J. D. Beasley, ”Fracture reduction using a biodegradable material, polylactide,” Oral Surg., 29, 393-397 (1971). 30. L. Getter, D.E. Cutright, S.N. Bhaskar, and J.K. Augsburg, “A biodegradable intraosseous appliance in the treatment of mandibular fractures,” J. Oral Surg., 30,344-348 (1972). 31. K.C. Pani, G. Gladieux, G. Brandes, R. K. Kulkarni, and E Leonard, “The degradation of N-butyl alpha cyanoacrylate tissue adhesive. 11,” Surgery, 63,481-489 (1968). 32. P. Christel, F. Chabot, J.C. Leray, C. Morin, and M. Vert, ”Biodegradable composites for internal fixation,” in Biomaterials 1980, G. D. Winter, D. E Gibbons, H. Plenk (eds.), Wiley, New York, 1982,pp. 271-280. 33. W. Korsatko, B. Wabnegg, H.M. Tillian, C. Braunegg, and R.M. Lafferty, ”Poly-D(-)-3-hydroxybuttersaure-ein biologisch abbaubarer Arzneistofftrager Lur Liberarationsverzorgerung 2,” Pharrn. Ind., 45, 1004-1007 (1983). 34. J. Vasenius, “Is n-butyl-2-cyanoacrylate a biocompatible coating material for biodegradable fracture fixation devices?,” Clin. Mater., 3, 133143 (1988). 35. R.A. Milch, D. P. Rall, J. E. Tobie, J. M. Albrecht, and G. Trivers, ”Fluorescence of tetracycline antibiotics in bone,” J. Bone Jt. Surg., 40-A,897910 (1958). 36. R. Schenk, ”Zur histologischen verarbeitung von unentkalkten knochen,” Acta Anaf., 60, 3-19 (1965). 37. J. Goldner, ”A modification of Masson trichrome technique for routine laboratory purposes,” Am. J. Puthol., 14, 237-243 (1938). 38. W. Bonfield, J. Bowman, A. Brandwood, C. Doyle, and D. Saunders, “Hydroxyapatite reinforced polyhydroxybutyrate as a biodegradable composite,” in Abstract book of European Congress in Biomaterials, Bologna, Italy, 1986,p. 193. 39. D. E Williams and N. D. Miller, “The degradation of polyhydroxybutyrate (PHB), in Abstract book of European Congress on Biomaterials, Bologna, Italy, 1986,p. 192. 40. S. Vainionpaa, K. Vihtonen, M. Mero, H. Patiala, P. Rokkanen, J. Kilpikari, and I?. Tormala, ”Biodegradable fixation of rabbit osteotomies,” Acta Orthop. Scand., 57, 237-239 (1986). 41. K. Vihtonen, S. Vainionpaa, M. Mero, H. Patiala, P. Rokkanen, J. Kilpikari, and P. Tormala, “Fixation of experimental osteotomies of
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the distal femur with biodegradable thread in rabbits,” Clin. Orthop., 221, 297-303 (1987). 42. N. Gammelgaard and J. Jensen, ”Wound complications after closure of abdominal incisions with Dexon or Vicryl. A randomized double-blind study,” Acfu Chir. Scand., 14% 505-508 (1983). Received September 20,1989 Accepted May 29,1990
coated with slowly absorbable polymers Jarkko Vasenius,* Seppo Vainionpaa, Kimmo Vihtonen, Matti Mero: Antero Makela, Pertti Tormala) and Pentti Rokkanen Department of Orthopaedics and Traumatology, Tiiolo Hospital, Helsinki University Central Hospital, Topeliuksenkatu 5 SF-00260 Helsinki; 'The College of Veterinary Medicine, Hameentie 57, SF-00550 Helsinki; fBiomaterials Laboratory, Institute of Plastics Technology, Tampere University of Technology, PO. Box 527, SF-33101 Tampere, Finland A total of 150 osteotomies of rabbits' distal femur were fixed with absorbable selfreinforced polyglycolide (SR-PGA) rods coated with slowly absorbable polymers. In order to reduce the degradation rate of the SR-PGA construction rods were coated with n-butyl-2-cyanoacrylate, polydioxanone (PDS), poly-beta-hydroxybutyrate (PHBA), or poly-1-lactide (PLLA).Biocompatibility and the rate of biodegradation were evaluated in histological, histomorphometric, microradiographic, and oxytetracycline labeling studies. PDS, PHBA, and PLLA coatings showed good biocompatibility although there were scattered fluid accumulations around the
rod in each group. Cyanoacrylate coating seemed to inhibit cartilage regeneration and cause the formation of considerable amount of connective tissue around the implant. The PGA core of the rod had totally degraded in 24 to 36 weeks in each coating group. Cyanoacrylate and PDS coatings were not detectable after 6 weeks while PHBA and PLLA coatings were still observed after 48 weeks. There were five (14%) non-unions in the cyanoacrylate coating group, one (3%)in the PHBA coating group caused by a purulent infection and none in the PDS and PLLA coating groups.
INTRODUCTION
Absorbable polyglycolide (PGA) osteosynthesis implants were introduced by Schmitt and Polistina 1969.' PGA is widely known as the first synthetic absorbable suture material (Dexon).' Its biocompatibility and biodegradation in soft and hard tissue has been proven in several experimental and clinical Rokkanen et al. presented the first clinical series in which absorbable PGA/ PLA rods and sutures were compared with conventional metallic screws and plates in the fixation of malleolar fractures.' In both groups the results were equally favorable, but unlike the absorbable implants metallic implants had to be removed operatively after the fracture had healed. During developing ab*To whom correspondence should be addressed. Journal of Biomedical Materials Research, Vol. 24, 1615-1635 (1990) 0 1990 John Wiley & Sons, Inc. CCC 0021-9304/90/121615-21$04.00
VASENIUS ET AL.
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sorbable osteosynthesis implants about 2000 animals have been operated in experimental trials and more than 900 patients have been treated with absorbable osteosynthesis implants at our department so far. The excessive rigidity of the fracture fixation performed with the metallic implants causes osteoporosis and atrophy of the cortical bone underneath the implant?" This may lead to secondary fractures after the removal of 0steosynthesis implants especially from diaphyseal bone. The elasticity modulus of PGA implants (10-15 GPa)'' is close to that of cortical bone (6-20 GPa)"12 which diminishes the risk of such disadvantages. The initial bending and shear strength of SR-PGA rods (200 5 25 MPa and 350 k 50 MPa)13,'4have been superior to other absorbable devices presented in the 1iterat~re.l~'~ However, PGA is the most hydrophilic absorbable polymer and SR-PGA rods lose their mechanical properties in 6-8 weeks in 37°C distilled water and in 5-6 weeks in the subcutis of rabbits.I4For this reason the clinical use of SR-PGA osteosynthesis implants has been limited to the fixation of fractures and osteotomies which heal relatively fast and where the fixation site is not exposed to hard stress during fracture healir~g.~,'~ Hydrolysis in aqueous environment is considered the main pathway of degradation of PDS, PGA, PHBA, and PLA, but the initiation of this process could be enhanced by enzymatic activity at least with PGA and PLA.'9-21The end products of PDS, PGA, PHBA, and PLA degradation are excreted in exhaled carbon dioxide (COJ and in urine.2'22-25 N-butyl-2-cyanoacrylate is degraded into formaldehyde and alkylcyanoacetate.26Polydioxanone (PDS), poly-beta-hydroxybutyrate(PHBA) and polylactide (PLA) have shown good biocompatibility in soft and hard t i s ~ u e . ~ ~They ~ ' - ~all " degrade slower than PGA .23,31-33 N-butyl-2-cyanoacrylate is better known as an absorbable tissue adhesive (Histoacryl).Unlike its monomer form n-butyl-2-cyanoacrylate polymer has been well tolerated by bone The aim of the present study was to evaluate the biocompatibility and absorption rate of the SR-PGA rod coated with four different slowly absorbable polymers after implantation in cancellous bone of rabbit. Rods were coated in order to decrease their degradation rate. EXPERIMENTAL METHODS
Implant materials Cylindrical self-reinforced polyglycolide (SR-PGA) implants were manufactured by partially melting and sintering commercial PGA sutures (Dexon " S size USP 2, Davis Geck, Gosport, U.K.) at temperatures 205"C-232"C at elevated pressure. In the coating process SR-PGA rods were five times briefly immersed in 55°C solutions of chloroform containing 0.75% and 1.5% PDS (Ethicon, Norderstedt, FRG), PHBA (Marlborough Biopolymers, L td., Cleveland, U.K.) or poly-1-lactide (PLLA; CCA Biochem b.v. Gorinchem, The Netherlands). After the immersion the chloroform was evaporated leaving a
+
SR-PGA IMPLANTS COATED WITH POLYMERS
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30-40-pm thick continuous polymer layer on the rod. A cyanoacrylate coating of equal thickness was laid down at room temperature by immersing the rod briefly in n-butyl-2-cyanoacrylate monomer (Histoacryl blau, B. Braun Melsungen, West Germany) which polymerized on the rod. The thickness of the coating was evaluated by measuring the diameter of the rod before and after the coating process. The nominal diameter of the coated rod was 3.4 mm. Animal experiments and preparation of specimen A total of 150 adult rabbits of both sexes weighing 2400-4400 g were operated on. Rabbits received 0.5 mg/kg atropine (Atropin) subcutaneously as premedication and they were anesthetized with subcutaneous medetomidine (Domitor) 0.3 mg/kg, ketamine (Ketalar) 0.4 mg/kg, and diazepam (Diapam) 0.2 mg/kg. They were also injected subcutaneously 150000 IU procaine penicillin (Procapen) as infection prophylaxis. The right hind leg was shaved and scrubbed with antimicrobial solvent (Neo-Amisept).An anteromedial incision was made and patella was dislocated laterally. A transversal osteotomy was made in the cancellous bone of distal femur with a circular saw. A longitudinal drill channel, 3.4 mm in diameter and 2.5 mm long, was made in the intercondylar area. The osteotomy was fixed with coated PGA implant applied in the drill channel. The fixation was secured with 2-3 USP size 0 PGA sutures (Dexon, Davis + Geck, Puerto Rico) running through transversal drill channels made on the both sides of the osteotomy (Fig. 1 A,B). Openings were closed with PGA sutures and rabbits were returned to their cages and they received regular laboratory animal diet and water. No external support was used. The follow-ups and the number of rabbits followed is presented in Table I. Rabbits were sacrificed with an overdose of f luanizone (Hypnorm) and an intravenous air embolism. Three days before sacrifice they were injected with 50 mg/kg oxytetracycline (OTC; Terramycin) for fluorescence After sacrifice both femurs were exarticulated, dissected, and radiographs were taken in anteroposterior and lateral views. Any signs of infection or other complication were visually controlled. The distal portions of both femurs were fixed in a series of ethanol immersions with rising concentrations (70-99%) and then embedded in methylmetha~rylate.~~ The intact femurs’ served as control specimens. Longitudinal sections, 5 pm thick, were cut in frontal plane with microtome (Jung, Polycut S, 1983) and stained by using Mason-Goldner’s method for histological and histomorphometrical evalua t i ~ nFor . ~ OTC-f ~ luorescence and microradiographic studies longitudinal sections in frontal plane, 80 p,m thick, were prepared with saw microtome (Leitz 1600). In the evaluation of OTC-fluorescence the intensity of fluorescence was graded from 0 to 3 and the fluorescence index was calculated by subtracting the intensity of the intact control specimen from the operated one. In microradiography Kodak Professional film SO 343 (Eastman Kodak, Rochester, NY) and 50 kV, 9 mA, 12 min exposure time and 29.5 cm film-focus were used. In quantitative histomorphometric analysis, a light microscope, (Leitz Wezlar,
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.*imp1 ant
.. s u t u r e
(b) Figure 1. (a) Schematic frontal view of distal femur showing horizontal osteotomy, implant, and the suture pulled through drill channels which were drilled at the proximal and distal aspects of the osteotomy. (b) Side view of distal femur showing osteotomy, drill channels, the implant, and the suture.
Ernst Leitz Co., Wetzlag, FRG) linked via a video camera to a semiautomatic computerized analysis system, MOP-Videoplan (Kontron, Munich, FRG) was used. A magnification of x16 was used and the magnification of the computer itself was ~ 2 5 0 The . microscopic field was displayed on TV screen of the computer and measurements were performed by using a digitizer tablet and cursor linked to it. The area of cartilage at the entry of the drill channel was measured to analyze cartilage regeneration. The areas of connective tissue, trabecular bone and new bone around the implant, as well as the implant area were measured from three consecutive fields along the implant (Fig. 2). TABLE I Plan of Experimental Studies on Rabbits Number of rabbits followed up (weeks) Implanted SR-PGA rod
1
3
6
12
24
36
48
C yanoacrylate-coated PDS-coated PHRA-coated PLLA-coated
5 5 5 5
5 5 5 5
5 5 5 5
5 5 5 5
5 7 5 5
5 8 5 5
8 5 6 6
38 40 36
20
20
20
20
22
23
25
150
Total ~
Totalnumber of rabbits
36
SR-PGA IMPLANTS COATED WITH POLYMERS
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Measured parameters A:
cartilage connective tissue implant new bone trabecular / subchondral bone
B-C: connective tissue implant new bone trabecular bone
D:
callus bone connective tissue trabecular bone
Figure 2. Schematic frontal view of the right femur. The histomorphometric measurements were performed in four standardized areas (A-D). The left femur served as control and was studied in the same manner.
The area of callus bone, connective tissue, and trabecular bone in the medial condyle at the level of osteotomy were measured to analyze the consolidation of the osteotomy. In statistical analysis regression analysis and x2test were used. P value less than 0.05 was considered statistically significant. RESULTS
Macroscopic studies The implants were radiolucent and drill channels for the rod were difficult to detect. However, channels drilled for the sutures were still detectable 48 weeks after operation. Radiographs showed a bony union of osteotomies in 6 weeks, but the osteotomy was partly detectable up to 24 weeks in each group. A Failure of fixation with a displacement of fragments was found in five (14%)rabbits in the cyanoacrylate coating group (24 and 48 weeks after operation) and in one rabbit in the PHBA coating group (6 weeks after operation). The latter was due to a purulent arthritis. A purulent arthritis was detected in two other operated knee joints but the osteotomies had consolidated normally in those cases. Six rabbits had to be sacrificed prior the end point of the follow-up period due to postoperative respiratory infection or trauma. Histological and microradiographic studies
One week Two out of five osteotomies treated with cyanoacrylate or PHBA coated rods and three out of five osteotomies treated with PDS or PLLA coated rods showed a bony union in histological and microradiographic studies. Most of
VASENIUS ET ALL.
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the coatings had detached from the rod and strips of coating material were found at the entrance of the drill channel (Fig. 3). Early signs of biodegradation of PGA core were found only in the cyanoacrylate coating group. PLLA coating had retained the best on the rod. A strong new bone formation was found in the metaphysis, in the periosteum, and around the osteotomy. A thin layer of granulation tissue covered the entrance of the drill channel in each group.
Three weeks All osteotomies, except one that was treated with PHBA coated rod, showed a trabecular bone union in histological and microradiographic studies (Fig. 4).A strong new bone formation was found around the osteotomy, in the periosteum and in the metaphysis. A thin layer of new bone had initiated to grow on the rod in each group. More granulation tissue, some cartilage, and new bone had grown at the entrance of the drill channel. Early signs of degradation of the PGA core were detected at the periphery of the rod especially where the coating had been detached. Most of the coatings had detached from the rod, but one or two samples of each rod type had an entire coating. Scattered small fluid accumulations with phagocytizing macrophages were found attached to the rod in each group.
Figure 3. Curly strips of PLLA coating (*) detached from the PGA implant (I) at the entry of the drill channel one week after operation. (MassonGoldner, original magnification ~ 1 0 0 . )
SR-PGA IMPLANTS COATED WITH POLYMERS
(e)
Figure 4. A bony union of the osteotomy partly through external callus formation and partly through trabecular bone formation was found three weeks after operation. Note the formation of new bone around the implant (I). (Masson-Goldner, original magnification X5.)
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Six weeks All osteotomies except one had healed and were difficult to locate histologically and microradiographically. One fixation with a PHBA coated rod had failed due to an infection. Cyanoacrylate and PDS coatings were no longer found on rods. PHBA and PLLA coatings were observed, but they were mostly detached from the rod. The more the rod had coating attached the less it had degraded (Fig. 5).Macrophages with PGA in their cytoplasm were found inside and around the degrading implants in two samples in the cyanoacrylate coating group. In each group most of the rods had a lining of new bone on them. A thin layer of osteoid and connective tissue had formed between the rod and new bone. No signs of delayed inflammation reaction or foreign body reaction were observed. 12 weeks
All osteotomies had healed partly through trabecular bone union and partly through external callus formation. C yanoacrylate coated rods had degraded into a homogenous, liquid PGA mass. Reinforcing PGA fibers with granulation tissue intruding between them were observed in the other groups (Fig. 6). PGA core had degraded faster in the subchondral area than in the proximal metaphysis, Two PDS coated rods showed small expansions toward bone tissue or medullary cavity, which could be an early sign of sinus forma-
Figure 5. An undamaged PLLA coating (+) and new bone (B) on the PGA implant (I) showing only early signs of degradation 6 weeks after operation. (Masson-Goldner, original magnification ~ 2 0 . )
(e)
SR-PGA IMPLANTS COATED WITH POLYMERS
1623
Figure 6. Granulation tissue intruding between degrading PGA fibers (I) 12 weeks after operation. (Masson-Goldner, original magnification x40.)
tion (Fig. 7). Several macrophages were found in these fluid accumulations indicating only a nonspecific inflammation and phagocytosis of polyglycolide debris. New bone formation was found in the drill channel at the level of the osteotomy and at the entrance of the drill channel, that was still filled with connective tissue and some cartilage. At this follow-up the amount of bone and connective tissue around the implant was the highest. 24 weeks
Two osteotomies treated with cyanoacrylate coated rods showed a pseudoarthrosis, while all the other osteotomies had healed uneventfully. There were only remnants of PGA core found at the implant site, which was filled with granulation and connective tissue. There were two exceptions in the PHBA and PLLA coated rods with solid PGA fibers left and an almost entire coating around the implant. PHBA and PLLA coatings were still found at the implant site and bone tissue had grown on both sides of PHBA coating. The implant site was covered with bone or connective tissue. Cartilage and connective tissue filled still the narrow entrance of the drill channel. No signs of foreign body reaction or prolonged inflammation were observed. 36 weeks All osteotomies had healed uneventfully. PHBA and PLLA coatings were still present at the implant site. One rod with an undamaged PHBA coating
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Figure 7. An exceptionally fast degraded PDS coated SR-PGA implant and a fluid accumulation with a connective tissue lining (+) expanding into the bone marrow was detected 12 weeks after operation. Note the considerably faster degradation of the implant in the metaphysis than in the marrow cavity. (Masson-Goldner, original magnification x5.)
SR-PGA IMPLANTS COATED WITH POLYMERS
1625
had degraded exceptionally slowly while the rest of the rods were practically totally degraded. Bone and connective tissue lining around the implant site had been absorbed and shrunk toward the metaphysis (Fig. 8). There was still a gap in the bone structure at the entry of the drill channel which was filled with connective tissue and cartilage. One sample in the PDS coating group showed a foreign body reaction with dense focuses of lymphocytes. 48 weeks Three samples in the cyanoacrylate coating group showed a pseudoarthrosis with a displacement of one or both condyles. All the other osteotomies had healed uneventfully. PGA had been totally absorbed in each sample. Strips of PHBA and PLLA coating were still observed among bone and connective tissue (Fig. 9). The entrance of the drill channel was filled with bone iq a third of samples and with cartilage and connective tissue in the others. The trabecular bone tissue around the implant site resembled intact bone. Bane layer and connective tissue around the implant had been absorbed or immigrated toward the metaphysis in all groups except in the cyanoacrylate coating group where the amount of connective tissue around the implant remained high. The regeneration of cartilage had advanced well in all groups except in the cyanoacrylate coating group. Bone remodeling was still in progress.
Figure 8. A considerable amount of connective tissue (C) and regular bone marrow replaced the cyanoacrylate coated SR-PGA rod 36 weeks after operation. (Masson-Goldner, original magnification x 5.)
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VASENIUS ET AL
Figure 9. 48 weeks after operation remodeling was almost finished. Dense connective tissue and regular trabecular bone was found at the distal implant site and at the entry of the drill channel Note the strips of PLLA coating (+) in the connective tissue. (Masson-Goldner, original magnification ~5 and ~ 2 0 . )
(e).
1627
SR-PGA IMPLANTS COATED WITH POLYMERS
Histomorphometrical studies In each coating group the rod area had increased about 10% between the first and the third week (Fig. 1OA). In the PDS and PLLA coating groups the rod area started to decrease after 3 weeks and after 6 weeks in the cyanoacrylate and PHBA coating groups. At 12 and 24 weeks the area of PHBA coated rods was significantly larger compared to the rest of the rods ( p < 0.01). At 36 weeks the area of all implants, except one PHBA coated rod that was deleted from calculations, had decreased to zero. The area of connective tissue around the implant was largest between 12th and 36th weeks post implantation (Fig. 10B). In the cyanoacrylate coating group the amount of connective tissue remained high while it decreased close to zero in all the rest of the groups. The amount of cartilage at the entry of the drill channel returned close to that of the intact control side in all groups except in the cyanoacrylate coating group (Fig. 1OC). The amount of new bone in the medial callus reached the highest value three to 6 weeks after operation and de-
Implant area (rnm2, 30 Qanoacryiate coated 25
T
i
L
PDS coated PHBA coated
20
PLLA coated 15
10 1 5
0
I
I
1
3
6
12
24
36
48
Implantationtime (weeks) (a)
Figure 10. The histomorphometric analysis showed that the area of all rod types increased during the first 3 to 6 weeks and decreased to zero in 24 to 36 weeks (a). The amount of connective tissue around and in the implant reached its top between 6th and 12th weeks after operation and then started to decrease in all groups except in the cyanoacrylate coating group where the largest amount of connective tissue was found 36 and 48 weeks after operation (b). The amount of cartilage tissue at the entry of the drill channel was significantly smaller in the cyanoacrylate coating group than in the other groups (c). (* = cyanoacrylate vs. other coatings, p < 0.01. ** = PHBA vs. other coatings, p c 0.01).
VASENIUS ET AL.
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Connective tissue area (rnd) 25
20
15
10
5
0 1
3
6
12
24
Implantation time (weeks) (b)
2 Cartilage area (mm ) 4
3
2
1
0
Figure 10. (continued)
creased with remodeling of the bone. The amount of trabecular bone around the implant and did not show any significant differences between follow-up periods or coatings.
1629
SR-PGA IMPLANTS COATED WITH POLYMERS
Oxytetracycline fluorescence labeling studies The results of OTC fluorescence labeling studies are presented in Fig. 11 AD. Fluorescence in the operated specimen showed the highest intensity and thus strong new bone formation during the first 6 weeks in the periosteum, at the osteotomy, and in the trabecular bone, while at the entry of the drill channel for the implant it was lower than that of the intact specimen during the first 3 weeks increasing toward the 12th and 24th week, indicating rather late bone remodeling at that area. DISCUSSION
Diverging results of the degradation rate of PGA in bone tissue have been presented. Cutright et al. presented that only 17% of the PGA implanted in bone tissue had degraded in 31 weeks. C-14 labeling studies showed that 2.7% of the fast-cured PGA implant remained after 5 months, while 29.5% of the slow-cured PGA implant was left after 9 month^.^ Vainionpaa presented that only remnants of injection molded PGA implant were detected 12 weeks after implantation in cancellous bone.' In the present study the cyanoacrylate, PDS, and PLLA coated SR-PGA implants had degraded in 24 to
Periosteum Fluorescence 3,5
I
I Cyanoacrylate coated
31-
PDScoated
0 PHBAcoated PLLAcoated
?
3
6
12
24
36
48
Follow-up (weeks) (a)
Figure 11. Results of the OTC fluorescence studies. The intensity of OTC fluorescence was graded from 0 to 3 and the fluorescence index was calculated by subtracting the intensity of the intact control specimen from the operated one (0 = no fluorescence, 1 = weak fluorescence, 2 = moderate fluorescence, 3 = strong fluorescence).
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VASENIUS ET AL.
Osteotomy Fluorescence Cyanoacrylatecoated PDScoated
c]PHBAcoated
1
3
6
12
24
36
48
Follow-up (weeks) (b)
Subchondral bone Fluorescence 2
1
1
3
6
12
24
cyanoacrylate coated
36
b
48
Follow-up (weeks) (4 Figure 11. (continued)
36 weeks and PHBA coated implants in 36 weeks with one exception. At 12 to 24 weeks PHBA coated rods showed significantly slower degradation, which may be due to slightly better but still insufficient adhesion between PHBA coating and the SR-PGA core.
SR-PGA IMPLANTS COATED WITH POLYMERS
1631
Entry of the drill channel Fluorescence
Follow-up (weeks} (4 Figure 11. (continued)
Compared to the other three coating groups a higher failure rate of fixation of the osteotomy, a larger amount of connective tissue around the implant, and the lesser amount of cartilage at the entry of the drill channel were observed in the cyanoacrylate coating group. These adverse effects of cyanoacrylate coating may be due to its degradation product formaldehyde which is a toxic material and they make doubtful the biocompatibility of cyanoacrylate coating. In the present study strips of PHBA and PLLA coating were detected at the implant site still 48 weeks after implantation, which confirms the very slow degradation of PHBA and I'LLA.3233,38Some authors doubt about the biodegradation of PHBA.39 An identical fixation method with the present study was used by Vainionpaa et a1.40Implants used in their study were injection molded PGA rods without fiber reinforcement or coating. In their series all osteotomies healed uneventfully and the implants were replaced by bone tissue in 12 weeks. Vihtonene et al. showed that 71% of distal femoral osteotomiesfixed with pure PGA threads healed ~neventfully.~'Fixation of such osteotomies with pure I'GA rod has not been studied. In the present study 86% of the osteotomies fixed with cyanoacrylate coated rods showed undisturbed healing, while in the PDS and PLLA coating groups all osteotomies healed normally. In the PHBA group there was one non-union caused by a purulent infection, while the rest of the samples showed an uneventful healing. In the present study the area and thus the volume of the implant enlarged during the first 3 to 6 weeks. Scattered fluid accumulations and some expansions of degrading PGA toward the bone tissue were observed around the
1632
VASENIUS E’T AL.
implant in each group. These may be early signs of transient sinus formation, a phenomenon which has been reported in clinical studies on PGA and PGAI PLA sutures and PGA PGA is a very hydrophilic polymer and it absorbs fluids and expands as it degrades. This causes pressure on the surrounding tissues and a sinus may develop to the weakest point of the surrounding tissue. When the fixation of a fracture is concerned this would be the soft tissue around the mouth of the drill channel and the soft tissue around it. A transient sinus formation has never been reported in experimental studies on PGA rods. Unlike most clinical series the entry of the drill channel for the PGA rod has located intra-articularly in experimental studies. The joint capsule can resist hydrostatic pressure much better than scar tissue. Also the synovial tissue can absorb intra-articular fluid very well thus lowering the hydrostatic pressure in the capsule. According to the present study the degradation of coated SR-PGA rods takes 24-36 weeks which is 12-24 weeks longer than it takes injection molded PGA implants to degrade in cancellous bone tissue.6This difference seems to be mainly due to the self-reinforced structure and not due to the coating, because the adhesion between the PGA implant and any of the coatings used in the present study was insufficient for the implantation of coated rods in bone tissue. The majority of coatings had cracked and peeled at the implantation. Ethylene oxide sterilization may further weaken this adhesion. In our earlier study slowly absorbable coatings were able to improve statistically the strength retention of SR-PGA rods in the in vitro but not in the in vivo environment^.'^ The results of the present and our earlier studies have shown that slowly absorbable coatings which have a chemical structure which differs from that of the self-reinforced core can not provide major improvements to absorbable osteosynthesis implants. This study was supported by grants from the Foundation for Orthopaedic and Traumatology in Finland, from the Paulo Foundation, and from the Academy of Finland. We thank the following persons for technical assistance: Ms. H. Kuisma, Mr. J. Laiho, Mr. M. Tamminmaki, and Mr. J. Mikkola. The Finnish national guidelines for the care and use of laboratory animals has been observed in the present study.
References 1. E. E. Schmitt and R. A . Polistina, ”Surgical sutures,” U.S. Pat. No. 3 463 158,1969. 2. E. J. Frazza and E. E. Schmitt, ’A new absorbable suture,” 1. Bimed. Mater. Res. Symp., 1, 43-58 (1971). 3. J.B. Herrmann, R. J. Kelly, and G . A . Higgins, “Polyglycolic acid sutures,” Arch. Surg., 100, 486-490 (1970). 4. H. Dardik, I. Dardik, and H. Laufman, “Clinical use of polyglycolic acid polymer as a new absorbable synthetic suture,” Am. 1. Surg., 121, 656-660 (1971).
SR-PGA IMPLANTS COATED WITH POLYMERS 5. 6.
7. 8.
9. 10.
11. 12. 13.
14.
15.
16. 17.
18.
19. 20. 21.
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the distal femur with biodegradable thread in rabbits,” Clin. Orthop., 221, 297-303 (1987). 42. N. Gammelgaard and J. Jensen, ”Wound complications after closure of abdominal incisions with Dexon or Vicryl. A randomized double-blind study,” Acfu Chir. Scand., 14% 505-508 (1983). Received September 20,1989 Accepted May 29,1990
