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Biomechanical and Allergological Characteristics of a Biodegradable Poly(D,L-Lactic Acid) Coating for Orthopaedic Implants

Posted on: Sunday, 7 August 2005, 03:01 CDT

Abstract

A poly(D,L-lactic acid) surface coating (PDLLA) has been developed to optimize interactions at the implant-tissue interface. Mechanical and allergological characteristics were evaluated in the present study to elucidate possible indications and limitations prior to clinical application. Implants of stainless steel and Ti- 6A1-4V and Co-Cr-Mo alloys were coated with PDLLA, and mechanical stability was studied during intramedullary implantation into rat and human cadaver bones and during dilation of coronary artery stents. Elongation resistance was examined on AlMgSi alloy specimens. Furthermore, proliferation of peripheral blood mononuclear cells of nickel-allergic donors and controls and interleukin-4 and interferon-γ levels were measured in the presence of coated/uncoated implants and after stimulation with phytohemagglutinin or NiSO^sub 4^. PDLLA remained stable on the implants with a minimum of 96% of the original coating mass and tolerated lengthening of at least 8%. Further lengthening was followed by microcracking and cohesive failure within the coating. PDLLA exerted no suppressive effect upon spontaneous and pan-T-cell mitogen inducible T-cell proliferation. Furthermore, specific proliferation to nickel in cells of nickel-allergic blood donors and production of interleukin-4 and IFN-γ were not influenced by the coating. PDLLA coating proved high mechanical stability on different orthopaedic implants and did not influence in vitro T- cell reactivity towards specific biomaterials.

2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: PDLLA; Bonding strength; Shear strength; T-cell proliferation; Nickel allergy

Introduction

Development of biomaterials and their application in the medical field has developed into an essential part of modern medicine. However, biomaterial-associated complications like allergological and foreign body reactions, implant loosening and infection still limit the application of biomaterials in daily clinical practice. A crucial factor for successful implant integration is the interaction between living tissue and the biomaterial at the implant-tissue interface [8,10].

One strategy to improve implant integration is the modification of well-established biomaterials by means of surface coating. A biodegradable poly(D,L-lactic acid) surface coating (PDLLA) can improve interactions at the interface by means of a locally acting drug-delivery system. Drugs incorporated into the biodegradable polymer are released continuously over a period of several weeks and may help to reduce complication rates [6,7,21]. PDLLA can be applied to virtually any stable implant resulting in a dense and regular surface coating. The coating offers an exciting tool to improve implant integration and fracture healing by combining the biodegradable polymer with insulin like growth factor-I (IGF-I) and transforming growth factor-betal (TGF-β1).

Schmidmaier et al. demonstrated significantly improved fracture healing in vivo after application of PDLLA-coated intramedullary rods [18]. These results were most evident with the use of integrated growth factors; however, even pure polymer improved bone consolidation, demonstrating its excellent biocompatibility. Accelerated bone integration of PDLLA-coated implants has been observed in various orthopaedic applications [17-20]. Furthermore, reduction of bacterial colonization was achieved by integrating antibiotics into polymer coatings [6,7,15]. Similar antibacterial principles have also been described; for example, gentamicin has been successfully integrated into a polylactic-co-glycolic acid coating [16]. Excellent antithrombogenic characteristics have been shown for PDLLA, and reduction of restenosis after stenting in coronary artery disease could be demonstrated in vivo due to inhibition of neointima formation by antithrombogenic modification of the surface coating [1,11]. Individual combinations of required drugs in custom-tailored surfaces could significantly reduce biomaterial-associated complications, and consequences on clinical outcome and financial savings seem very promising.

Little is known, however, about the mechanical stability and both allergological and immunological characteristics of the PDLLA surface coating. Such information is important in choosing proper indications for the clinical field. The purpose of this study was to examine the mechanical properties of the surface coating in clinically oriented models. Furthermore, T-cell mediated immune response towards coated and uncoated metallic implants was investigated.

Table 1

Sample groups and preparations for the mechanical and allergological tests

Materials and methods

Resomer R203 (Boehringer Ingelheim, Ingelheim, Germany), a polymer of D,L-lactic acid with a molecular weight of 29,500 Da was used for implant coating. The polymer, as a raceme, consists of equal parts of the D- and L-enantiomer. In brief, implant coating was performed by solvent casting after dissolution of the polymer in organic solvents. 133.3 mg of PDLLA were dissolved per milliliter of ethyl acetate, and the specimens were coated by two consecutive dip- coating procedures. The solvent was then allowed to evaporate at room temperature (21 C) for 24 h. Methylviolet was incorporated into the polymer as a model drug in a concentration of 0.1% (w/w). An overview of sample preparations and test procedures is given in Table 1.

Mechanical stability

PDLLA adhesion to Kirschner wires (K-wires)

Ten commercially available K-wires (Synthes, Umkirch, Germany) made of stainless steel (according to ISO 5832-1) and K-wires made of Ti 6AMV (according to ISO 5832-11 and ISO 5832-3) with a diameter of 1.8mm served as substrate specimens and were coated with PDLLA. Total coating mass was determined with an electronic micro-balance (Sartorius AG, Gottingen, Germany, readability 0.01 mg). Then, without previous drilling, K-wires were incorporated proximally into rat tibiae (Sprague Dawley rats) as intramedullary rods. After explantation and removal of adherent bone and bone marrow, the loss of coating mass (denoted LCM) was determined gravimetrically.

PDLLA adhesion to orthopaedic endoprostheses

Stem modules of "MML" tumor endoprostheses (ESKA Implants, Lbeck, Germany) with a length of 120 mm and a diameter of 13 mm made of a Co-28Cr-6Mo alloy (ISO 5831-4, ASTM F75-01) were coated with PDLLA and a lipophilic dye (Sico Fettschwarz, 0.001% v/v). Photometric analysis was used for indirect measurement of coating loss during intramedullar implantation, because great weight difference between prosthesis and coating prevented gravimetrical determination. A calibration curve of the lipophylic dye was generated after photometric detection of serial dilutions at 591.5 nm with a conventional photometer (DU-600, Beckman, Palo Alto, CA). Based on the calibration curve, the total amount of PDLLA with integrated dye could be determined after complete dissolution from the metal surface by chloroform.

In the control group, PDLLA coating was completely removed from the endoprostheses without prior implantation and coating mass was analyzed photometrically (n = 7). Coated test group samples, however, were implanted under standardized conditions into the diaphyses of human cadaver femora after intramedullary reaming (n = 7). After X-ray control, the prostheses were explanted by means of osteotomy and carefully cleaned. Thereafter, the coating was completely detached by chloroform and quantified photometrically. In addition, three implant modules were coated with PDLLA and Coumarin 152A (LC 4810, Lambdaphysik, Gttingen, Germany), a fluorescent dye, to document the distribution of abrasive particles at the inner bone cortex after implantation under ultraviolet light.

Ducility and tension stress resistance of the PDLLA surface coating

Ductility of the coating was examined by light and scanning electron microscopy (JEOL 5900 SEM, JEOL Germany GmbH, Eching, Germany). In brief, flat alloy specimens made of Al-Mg-Si (according to DIN EN 573-3) with a rectangular cross-section of 20 2 mm (specimen length 130mm) and an elongation at fracture of 12-36% were used (Ran Vertriebs GmbH, Munich, Germany). The middle third of each specimen was coated evenly with PDLLA (Fig. 1). All experiments were performed using a universal testing machine (WOLPERT TZZ 707, Instron Wolpert, Darmstadt, Germany) with a 50 kN load cell (according to German and European industrial standard DIN EN 10002- 1) under displacement at a rate of 10mm/min. For better visualization, 0.1 % (w/w) inethylviolet was incorporated into the coating.

Non-destructive elongation of coated alloy specimens by 4% (group 1, n = 5) was defined with an extensomeler (Wolpert ZV 1069-01, Instron Wolpert). For elongation until failure of the alloy (group 2, n = 5), strain gauges (EP-080 -250BG-120Ω; Measurements Group, Raleigh, NC) with a maximum recordable elongation of 20% were used, and total tensile strength and elongation were determined. After elongation, the coatings were examined for debonding and failure of the coating by light microscopy and SEM.

PDLLA concentration in the coating solution and influence on coating stability

The influence of combined te\nsile and shear forces on coating stability was studied with coronary artery stents made of stainless steel (In Flow Dynamics, Munich, Germany). Stents were coated with 33.3 mg, 66.7 mg, and 133.3 mg of PDLLA per milliliter of ethyl acetate (n = 6 for each group). The influences of the polymer concentration on coating mass and adhesive stability were studied. After coating, the coronary artery stents were expanded lege artis with commercial balloon catheters (d = 3 mm, p = 8 atm), and LCM was determined with a precision micro-balance.

Fig. 1. Schematic illustration of test setup for elongation failure of PDLLA on Al-Mg Si metal plates.

Allergologicul characteristics

Cells find proliferation assay

Five nickel-allergic individuals (females, age 25-54 yr) and two non-allergic controls (males, age 32 and 35 yr) volunteered as blood donors. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll (Pharmacia, Freiburg, Germany) density centrifugation from 40 ml of heparinized venous blood and resuspended in AB-serum containing enriched RPMI 1640 medium (Pan Biotech, Aidenbach, Germany). Resuspended human PBMC were cultured (1 10^sup 6^/ml) with either complete culture medium alone or with the addition of phytohemagglutinin (PHA; 2.4 g/ml; Biochrom, Berlin, Germany) or nickel sulfate (NiSO^sub 4^; 10^sup -4^M and 10^sup -5^ M; Sigma- Aldrich, Taufkirchen, Germany) in culture plates (Corning, Corning, NY). In a parallel set of experiments, cell suspensions were cultured with additional presence of uncoated or PDLLA-coated metal disks (d = 15 mm) of stainless steel and Ti-6A1-4V alloy.

Assessment of cell proliferation and mediator production

After 5 days of culture in the presence or absence of the additional stimuli, ^sup 3^H-thymidine was added. To evaluate proliferation, cells were harvested after additional overnight incubation, and incorporated radioactivity was measured by liquid scintillation. The extent of proliferation was expressed as stimulation index (SI), calculated by the quotient of ^sup 3^H uptake in stimulated to non-stimulated cells.

From a parallel set of identically stimulated cultures, supernatants were collected at day 6 and analyzed for presence of interleukin-4 (IL-4) and interferon-γ (IFN-γ) by commercially available ELISA (BD Biosciences Pharmingen, Heidelberg, Germany).

Statistical analysis

Means and standard deviations were calculated. Statistical analysis was performed when applicable with Mann -Whitney test, with p < 0.05 considered significant.

Results

PDLLA adhesion to K-wires

Total coating mass on Ti-6A1-4V and stainless steel implants did not differ significantly (p > 0.05). After implantation of PDLLA- coated K-wires, LCM averaged 4.1 3.4% for stainless steel implants and 3.9 3.8% for Ti-6A1-4V (p > 0.05). Thus, about 96% of the PDLLA coating mass remained attached to the K-wires during intramedullary implantation. No adhesive failure between the coating and the substrate was observed, and LCM was mainly due to abrasion with cohesive failure within the PDLLA coating itself.

PDLLA adhesion to orthopaedic endoprostheses

Large area contact between the implant and the inner cortex of the femur was controlled radiologically and was confirmed for all implants. One experiment had to be repeated since a longitudinal fracture of the femur occurred during implantation and thus appropriate shear stresses at the implant-bone contact area were no longer guaranteed.

A mean of 116.00 18.65 mg of PDLLA was measured on the implants of the control group, whereas 111.82 18.33 mg could be detached from the implanted endoprostheses. Therefore, an LCM of 4.6% resulted from intra-osseous implantation into human femora. Evaluation under fluorescent light showed an even distribution of coating abrasion over the entire bone stock.

Ductility and tension stress resistance of PDLLA surface coating

Group 1: After 4% of elongation, no morphological changes could be observed in the coating. PDLLA showed neither cracks nor debonding from the metallic surface. The test strength necessary for elongation amounted to 261 2 MPa for the coated specimen, a value corresponding to the reference value of the uncoated alloy.

Group 2: Specimens of this group were elongated to the breaking point, and elongation at fracture averaged 7.8 3.0%. Ultimate tensile strength averaged 783 0.4 MPa. The coating had no influence on the mechanical properties of the aluminum specimens.

Elongation to failure did not cause changes in the coating or debonding of PDLLA (Fig. 2). All coatings remained unchanged in the area of the strain gauges up to a mean elongation of at least 7.8%. Adhesive failure could only be observed directly at the disruption zone, where regular microcracking perpendicular to the tension direction were detectable through SEM. An elongation of more than 7.8% must be assumed due to the clearly visible specimen constriction in this area (Fig. 2). PDLLA proved to behave elastically without brittle fracture or further macroscopic fissures in the coating.

Fig. 2. Schematic illustration of a coated aluminum specimen after elongation to fracture (upper left). SEM image of the undamaged PDLLA coating in the area of the strain gauge (lower left). Perpendicular microcracking in the fracture area with elongation >7.8% (upper right). Fracture zone through light microscopy: no debonding of the coating could be observed (lower right).

PDLLA concentration in the coating solution and influence on coating stability

Total coating mass on the coronary artery stents (Fig. 3) increased proportionally to polymer concentration in the organic solvent (p < 0.05). However, no correlation was found between the polymer concentration in the coating solution and coating stability during dilation (p > 0.05). Loss of surface continuity with adhesive failure was caused by debonding of macroscopically visible polymer particles in single samples. Debonding of larger particles led to the high standard deviations in the experimental groups (Fig. 4). However, more than 95% of the polymer remained on the implant after stent dilation.

Fig. 3. Coating mass on cardiovascular stents in relation to the polymer concentration in the volatile organic solvent.

Fig. 4. LCM after stent dilation in relation to the polymer concentration in the volatile organic solvent.

Reactivity to titanium materials

In the presence of both coated and uncoated titanium samples, spontaneous in vitro proliferation of human PBMC was not altered, and T-cell stimulation by PHA led to almost identical, marked proliferation. Upon addition of NiSO^sub 4^, cells of nickel- allergic individuals showed a dose dependent proliferative response. This was not hindered by the presence of both uncoated and PDLLA- coated titanium samples, but rather slightly facilitated in the presence of PDLLA (Fig. 5a). The extent of spontaneous (preexisting) IFN-γ-formation in vitro showed marked inter-individual variation. As expected, IFN-γ levels strongly increased upon PHA addition and were also enhanced upon NiSO^sub 4^ addition to cells of nickel-allergic individuals independent of the coating. IL- 4 production also remained unchanged in the presence of uncoated and coated titanium materials (Figs. 6a and 7a).

Reactivity to stainless steel materials

Cellular reactivity was assessed in culture medium with coated and uncoated steel materials and upon PHA stimulation in absence and presence of coated and uncoated steel materials. No inhibitory effect was found upon PHA-induced T-cell proliferation (Fig. 5b). Baseline proliferation in PBMC of both nickel-allergic and non- allergic individuals was slightly enhanced by the presence of steel materials (both coated and uncoated). Again, release of IFN-γ and IL-4 was not influenced by PDLLA coating (Figs. 6b and 7b).

Fig. 5. Proliferative response in vitro (indicated as stimulation index, SI) of human PBMC in the presence of culture medium alone and upon addition of pan-T-cell-stimulator phytohemagglutinine (PHA) or nickel sulfate (NiSO^sub 4^) with (a) PDLLA-coated (+; n = 7) or uncoated (-; n = 5) titanium disks, (b) PDLLA-coated (+; n = 5) or uncoated (-; n = 5) stainless steel disks. Shaded symbols = nickel- allergic individuals; open symbols = non-allergic individuals. NiSO^sub 4^ (4) = NiSO^sub 4^ 10^sup -4^ M, NiSO^sub 4^ (5) = NiSO^sub 4^ 10^sup -5^ M.

Discussion

Coatings applied to orthopaedic implants must provide high stability without changing the mechanical properties of the substrate. Various test methods have been developed to study coating stability [5,13,14,22, 25,26]. However, these in vitro tests mainly focus on solitary stresses like bending forces and tensile, shear, or compression loads without simulating the in vivo situation, where forces act in a complex manner and combined stresses predominate. Mechanical stability of the PDLLA coating was therefore investigated for specific applications in clinically oriented models. Short-term investigations were performed; long-term stability and drug release are subject to a complex interaction of influences, such as (partial) polymer degradation, implant loading, drug characteristics, and surrounding media conditions [2], and are preferably studied for the specific application under in vivo conditions. Degradation of the applied PDLLA polymer depends on the implantation site and the thickness of the polymer film and is generally degraded in the organism over several months [12].

Fig. 6. IL-4 production in vitro (pg/ml) of human PBMC in the presence of culture medium alone and upon addition of pan-T-cell- stimulator phytohemagglutinine (PHA) or nickel sulfate (NiSO^sub 4^, 10^sup -4^ M, 10^sup -5^ M) with (a) PDLLA-coated (+; n = 7) or uncoated (-; n = 5) titanium disks, (b) PDLLA-coated (+; n = 5) or uncoated (-; n = 5) stainless steel disks. Shaded symbols = nickel- allergic individuals; open symbols = non-allergic individuals.

F\ig. 7. IFN-γ production in vitro (pg/ml) of human PBMC in the presence of culture medium alone and upon addition of pan-T- cell-stimulator phytohemagglutinine (PHA) or nickel sulfate (NiSO^sub 4^, 10^sup -4^ M, 10^sup -5^ M) with (a) PDLLA-coated (+; n = 7) or uncoated (-; n = 5) titanium disks, (b) PDLLA-coated (+; n = 5) or uncoated (-; n = 5) stainless steel disks. Shaded symbols = nickel-allergic individuals; open symbols = non-allergic individuals.

Important limitations refer to the adaptability of test methods for specific coating materials. ASTM C-633-01, for example, is a standard test to evaluate adhesion and cohesion strength of inorganic coatings [3]. Bonding agents with epoxy resins and organic solvents are applied to fix the substrate coating to the loading fixture. Penetrating bonding agents may interact with biodegradable polymer coatings like PDLLA, invalidating mechanical stability or even dissolving the polymer. Insoluble excipients and integrated drugs also significantly influence mechanical stability of surface coatings. Influencing factors are particle size, morphology, concentration, and surface chemistry of the integrated substances [4]. Stability testing is therefore recommended for every specific application.

Clinically oriented tests have been performed for different applications in the present study. Short-term mechanical testing was performed, as PDLLA has major indications in the early postoperative period with acceleration of bone healing and prevention of bacterial colonization [15,20]. PDLLA proved good stability on orthopaedic implants even during intramedullary implantation. Since abrasion of the coating was distributed evenly along the inner bone cortex, incorporated drugs would be 100% available at the action site. Abrasion occurred due to flaking of small polymer particles and did not differ significantly between stainless steel, Ti-6A1-4V, and Co- 28Cr-6Mo alloys. Coating thickness and bonding strength differences on different materials described by Schmidmaier et al. [21] were not observed in our study. This discrepancy may be due to the application of different organic solvents during the coating procedure, resulting in different polymer decomposition products after dissolution and consequently in different mechanical properties [4]. These aspects will be addressed in further studies.

Elongation by at least 7.8% did not cause any damage or debonding of the PDLLA coating. An elongation to fracture of at least 8%i is demanded for surgical Co-Cr-Mo alloys according to ISO 5832/4 and ASTM F75-01. PDLLA therefore proved suitable for coating of surgical implants like endoprostheses, intramedullary nails, and other fixation devices.

PDLLA also remained attached to coronary artery stents-dilated multiple times their original size-to more than 90%. Adhesive failure due to flaking of polymer particles was the typical failure mode, resulting in uncoated surface areas and loss of coating continuity. Interestingly, coating stability was independent of the original PDLLA concentration in the organic solvent. On the other hand, coating thickness could be modified by changing the polymer concentration in the coating solution, allowing adaptation for different clinical applications. PDLLA should therefore be applied to medical implants exposed to high deformation ranges only as a local drug delivery system, as complete sealing of the underlying biomaterial can not be guaranteed. Possible indications include tissues with minor perfusion, where systemic drugs do not reach therapeutic levels and locally released drugs can be effective for a long period. PDLLA coating also proved effective in vascular systems [1,11]; however, due to flushing of released drugs from the surface, drug levels decrease significantly faster than in less perfused tissues like bone.

Inexpensive implant materials with good mechanical properties but minor biocompatibility could be revaluated with surface coatings. Furthermore, the risk of allergic reactions might even be reduced. All implanted materials come in contact with circulating cells, e.g. lymphocytes and monocytes, and might thereby also induce and maintain a surrounding "immuno-incompetent fibro-inflammatory zone" [9]. Thus we wondered if PDLLA might alter lymphocyte reactivity in vitro e.g. could it influence the reaction of sensitized immune cells towards the underlying substrates or would it alter antigen- specific T-cell response. There was no suppressive effect upon spontaneous and pan-T-cell mitogen (PHA) inducible T-cell proliferation.

Since PBMC of nickel-allergic individuals often show specific (antigen-induced) proliferation in vitro upon stimulation by nickel [23], this model was used to further examine influence of coated and uncoated titanium materials upon antigen (nickel)-specific T-cell reactivity. However, specific proliferation to nickel in cells of nickel-allergic blood donors was neither enhanced nor suppressed in both conditions. Production of IFN-γ as characteristic mediator of delayed type hypersensitivity was also not influenced by PDLLA coating. Steel material did not impair mitogen-induced in vitro T- cell proliferation. In contrast with the restriction of a limited number of tested samples spontaneous in vitro proliferation tended to increase. Whether the extent of nickel release from these materials reaches the threshold for T-cell activation and thus contributes to this observation is the topic of ongoing studies. Microporous surface modifications have been previously found to induce IFN-γ production (unpublished data, Thomas et al.). In the case of PDLLA, no such changes were observed.

Taken together, PDLLA coating did not exert immunosuppressive or immunomodulatory effects upon in vitro T-cell reactivity. The influence of PDLLA coating upon release of potential allergens, e.g. nickel, chromium, or cobalt, from the respective material will be evaluated after a respective larger experimental series. With the restriction of naturally occurring inter-individual differences, assessment of in vitro T-cell reactivity appears useful as additional in vitro biocompatibility assay [24].

Biomechanical testing of biodegradable polymer coatings is challenging due to missing standards, and various clinically oriented models have been applied in the present work. PDLLA proved good stability and adhesion on orthopaedic implants even under high stresses, and demonstrated excellent elasticity up to an elongation of 8%. The in vitro data suggest that peri-implant T-cellular immunoreactivity is neither compromised nor changed to hyperreactivity in the presence of PDLLA coating. Further studies on metal release will show whether allergic reactions to implant constituents can be reduced by PDLLA coating.

Acknowledgment

This study was supported by a grant from the "Bavarian Research Cooperation for Biomaterials (FORBIOMAT)" of the Bavarian State Ministry of Sciences, Research and the Arts.

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Hans Gollwitzer a,b,*, Peter Thomas c, Peter Diehl a, Erwin Steinhauser a, Burkard Summer c, Sonja Barnstorf c, Ludger Gerdesmeyer a, Wolfram Mittelmeier d, Axel Stemberger e

a Klinik und Poliklinik fr Orthopdie und Sportorlhopdie, Technische Universitt Mnchen, Ismaninger Str. 22, 81675 Mnchen, Germany

b Abteilung fr Septische Chirurgie, Berufsgenossenschaftliche Unfallklinik Murnau, Prof.-Kntscher-Str. 8, 82418 Murnau, Germany

c Klinik und Poliklinik fr Dermatologie und Allergologie, Universitt Mnchen, Frauenlobstr. 9-11, 80337 Mnchen, Germany

d Orthopdische Klinik und Poliklinik, Universitt Rostock, Ulmenstr. 44145, 18055 Rostock, Germany

e Institut fr Experimentelle Onkologie und Therapiejorschung, Technische Universitt Mnchen, Ismaninger Str. 22, 81675 Mnchen, Germany

Accepted 26 January 2005

* Corresponding author. Address: Klinik und Poliklinik fr Orthopdie und Sportorthopdie, Technische Universitt Mnchen, Ismaninger Str. 22, 81675 Mnchen, Germany. Fax: +49 4140 7242.

E-mail address: h.gollwitzer@lrz.tu-muenchen.de (H. Gollwitzer).

Copyright Journal of Bone and Joint Surgery, Inc. Jul 2005

Source: Journal of Orthopaedic Research

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