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Initial biocompatibility studies of a novel degradable polymeric bone substitute that hardens in situ
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
ELSEVIER
JNITIAL BtOCOMPATJBJLJTY STUDJES OF A NOVEL DEGRADABLE POLYMERIC BONE SUBSTlTUTE THAT HARDENS IN SITU Steven Bennettl, Kevin Connollyl, Daniel R. LeeI, Ying Jiangl Dave Buck2, Jeffrey 0. Hollinger2, Elliott A. Gruskinl 1United States Surgical Corporation, 195 McDermott Ave. North Haven CT 06473 20regon H#ealthSciences University, 3181 SW Sam Jackson Park Rd. Portland, OR 97201-3098
ABSTRACT
Clinical management of osseous defects often requires bone grafts. The standard for treatment is autogenous bone harvested from sites such as either the iliac crest or the outer table of the calvaria. In addition to the problem of donor site morbidity and the limited supply of graft material, there is the additional operating time associated with harvesting procedures. A synthetic, bone graft substitute that can match the clinical performance of autogenous bone could alleviate these deficiencies. Therefore, a polymeric bone substitute was developed that consists of a four-armed star polymer of poly(dioxanone-co-glycolide) endcapped at each termini with a biocompatible lysine-based diisocyanate crosslinker. The polymer can be mixed with inorganic fillers such as either hydroxyapatite or tricalcium phosphate to fotm either injectable or moldable putty. The addition of a catalyst (for example, diethylaminoethanol and water) to the polymer produces a crosslinking reaction causing the combination to harden. This reaction is nontoxic, normo-thermic and can be performed in situ. During the course of the polymerization, carbon dioxide is liberated, producing an
interconnected porous network within the implant, suitable for bone ingrowth. This paper will describe a preliminary biocompatibility assay of the bone substitute.
INTRODUCTION
An effic,acious, resorbable, synthetic bone graft substitute should promote bone ingrowth and resorb in a predictable manner in register with new bone formation (for a recent review see 2). The
Address for correspondence and reprints: E. A. Gruskin, United States Surgical Corporation, 195 McDermott Ave., North Haven, CT 06473.
0 1996 by Elsevier Science Inc. All rights reserved.
101s
8756-3282/96/$15.00 PI1 S8756-3282(96)00130-5
102s
S. Bennett et al. Degradable polymeric
bone substitute
Bone Vol. 19, No. I, Supplement July 1996:101S-107s
capability for bone ingrowth into a porous implant is underscored by the bone-implant interface. This interface must prevent soft tissue intrusion that can inhibit bone growth. A rigid implant may not be suitably contoured and mortised into an irregular recipient bed. Therefore a moldable, or injectable material that hardens in situ and adapts to interface contours will have significant clinical utility. For example, Constanz and coworkers developed a liquid mixture of inorganic minerals that crystallizes into dahllite after introduction into fracture sites. The resulting micro-porous implant erodes from the surface and is replaced by bone in a coupled process (1). An alternative to a nonporous, erodable implant is to provide a porous scaffold for bone ingrowth. For example, Mikos and coworkers described a system that consists of a mineral component and unsaturated, linear polyesters that are polymerized in situ by a free radical-based reaction. The material includes 250-425 pm sodium chloride particles that dissolve, leaving behind a porous, polymer and mineral composite (6). The bone graft substitute we developed can be introduced as a putty to conform to the geometry of the bone defect. To create pores we used a non-toxic moiety that causes the release of carbon dioxide during the polymerization reaction. In addition to generating interconnected pores, the carbon dioxide causes expansion of the implant The result is a porous bone graft substitute with a seamless graftbone interface that allows bone ingrowth and prevents soft tissue prolapse.
Our novel polymer system that hardens in situ into a foam-like porous implant consists of a hydroxyl-terminated biodegradable polymer end capped with a reactive lysine-based diisocyanate moiety. To achieve a three dimensional crosslinked network, the polymeric base unit must have more than two reactive end-groups. Therefore, a “star polymer” ofp-dioxanone and glycolate was developed with four crosslinkable ends. Since the polymer was designed to be a liquid at room temperature, there is no need for a separate solvent to dissolve the reactants. Linked to each terminal hydroxyl group is a reactive crosslinker, similar to diisocyanate, but designed to be nontoxic. The result is a reactive “star polymer” primed to form an intricate three dimensional network upon polymerization. The polymer can be combined with either hydroxyapatite or tricalcium phosphate to form lactomer@ diisocyanate putty (LDIP). Prior to implantation, a catalyst is added to drive the crosslinking reaction. Since the diisocyanate group is used to synthesize polyurethane and polyurea, its crosslinking reactions have been thoroughly characterized (4). In this case, polymerization is catalyzed by water and a tertiary amine and carbon dioxide is liberated. Carbon dioxide release causes a foaming reaction that expands LDIP to fill voids in the bone defect. The reaction is complete in about ten minutes and the porous implant is retained at the recipient site through mechanical interlocking. In addition, the porous network has an architecture similar to bone, further enhancing the biomimetic effect of the bone substitute (FIG. 2).
In this report we describe the synthesis of LDIP and show an initial biocompatibility study. LDIP was polymerized in molds and implanted in the pectoralis muscles in rats. The implants were
Bone Vol. 19, No. 1, hpplement July 1996:101S-107s
S. Bennett et al. Degradable polymeric bone substitute
evaluated clinically and histologically at days 1,3,7, 14,21, and 42 for host tissue responses. Over the course of the study, the tissue responses to the implanted polymer were without unusual sequelae.
MATERIALS
& METHODS
Polymer Svnthesis The star polymer backbone of poly(dioxanone-co-glycolide) (PDO/G *) was prepared using 48.0 g pentaerythritol (Aldrich, Milwaukee, WI), 164.9 gp-dioxanone (United States Surgical Corp., Norwalk, CT), 24.1 g glycolide (United States Surgical Corp., Norwalk, CT), and 0.6 g stannous octoate (Brand NuLabs, Meriden, CT). The reactants were combined under nitrogen, heated to 90 oC for 60 hrs with stirring and then subjected to vacuum
. .. Biocompaublhty Sh.Q LDIP w,as prepared with either hydroxyapatite or p-tricalcium phosphate (10 pm particle size, Himed Inc, Gld Bethpage, NY). Standardized implants were formed by polymerizing LDIP in 3 mm diameter by !j mm cylindrical Teflon molds and the resulting porous rods were measured to confii uniformity. All implants were inserted surgically in the pectoralis muscles of rats following standard operating procedures (3). The rats were euthanized at days 1,3,7, 14,21 and 42 and the implant sites were examined clinically and histologically.
103s
104.9
S. Bennett et al. Degradable polymeric
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
bone substitute
II
I
0
+
KCl,
CbC
*
Iv
m
oc
TEA, 40 brs, RT
0
oc\
7
OCN V
FIG. 1: Schematic representation of LDI synthesis.
RESULTS
& DISCUSSION
Clinical examination did not reveal any signs of host incompatibility. Histologically, a transient inflammatory reaction occurred until day 3, at which time a minimal number of foreign body giant cells were observed. However, after the initial inflammatory reaction, the local response revealed a
Bone Vol. 19, No. 1, Supplement July 1996:1OlS-107s
S. Bennett et al. Degradable polymeric bone substitute
rich fibro-vascular sheath, without evidence of a sustained inflammatory reaction. The polymer implants were well integrated with the host tissue. In addition, there were numerous vascular buds that grew into several implants (FIG. 3 and 4). Importantly, concurrent with this process was a fibrovascular penetration of the implant, thus indicating degradation byproducts were not posing a toxic challenge.
FIG. 2: Scanning electron microscope ev&&n
of the interface between
The
bone-implant interface crosses vertically in the center of the image.
CONCLUSIONS
Preliminary biccompatibility data appear to indicate that LDIP does not elicit an adverse tissue response following an intramuscular implantation. The polymer surface appears to be an acceptable substrate for cell attachment. Any byproducts from the polymerization reaction do not cause significant toxicity and the degradation products are well tolerated. Additional experiments are planned for o’rthopic sites to assess the osteoconductive potential of this class of polymers for bone repair.
105s
106s
S. Bennett et al. Degradable polymeric
*y-to . FI Panel
* 1 i
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
bone substitute
.
. .
.
ralis muscle&.
A; day 3; Panel B; day7; PanelC; day 14; Panel D; day21; PanelE; day42.
A
g
. .
.
mtncalciump&e
in rat m
muscles: Panel A; day 3; Panel B; day 7; Panel C; day 14; Panel D; day 21; Panel E; day 42.
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
S. Bennett et al. Degradable polymeric bone substitute
REFERENCES
1) Constanz BR, Ison IC, Fulmer MT, Poser RD, Smith ST, VanWagoner M, Ross J, Goldstein SA, Jupiter JB, and Rosenthal DI. Skeletal Repair by in situ Formation of the Mineral Phase of Bone. Science 267: 1796- 1799; 1995. 2) Gazdag AR, Lane JM, Glaser D, and Forster RA. Alternatives to Autogenous Bone Graft: Efficacy and Indications. J. of the Amer. Acad. of Orthopaedic Surgeons 3: 1-8; 1995. 3) Katz RW, Hollinger JO and Reddi A.H. The funcional equivalence of demineralized bone and tooth matrices in ectopic bone induction. J Biomed Mater Res 27:239-245; 1993. 4) March J. Advanced Organic Chemistry. John Wiley & Sons, New York Chichester Brisbane Toronto Singapore, ~786; 1985. 5) Storey RF, Wiggins JS, Mauritz KA and Puckett AD. Bioadsorbable composites. II: Nontoxic, L-lysine-based poly(ester-urathane) matrix composites. Polymer Composites 14 :17-25; 1993. 6) Yaszemski MJ, Payne RG, Hayes WC, Langer RS, Aufdemorte TB and Mikos AG. The Ingrowth of New Bone Tissue and Initial Mechanical Properties of a Degrading Polymeric Composite Scaffold. Tissue Engineering I:41-52; 1995.
107s
ELSEVIER
JNITIAL BtOCOMPATJBJLJTY STUDJES OF A NOVEL DEGRADABLE POLYMERIC BONE SUBSTlTUTE THAT HARDENS IN SITU Steven Bennettl, Kevin Connollyl, Daniel R. LeeI, Ying Jiangl Dave Buck2, Jeffrey 0. Hollinger2, Elliott A. Gruskinl 1United States Surgical Corporation, 195 McDermott Ave. North Haven CT 06473 20regon H#ealthSciences University, 3181 SW Sam Jackson Park Rd. Portland, OR 97201-3098
ABSTRACT
Clinical management of osseous defects often requires bone grafts. The standard for treatment is autogenous bone harvested from sites such as either the iliac crest or the outer table of the calvaria. In addition to the problem of donor site morbidity and the limited supply of graft material, there is the additional operating time associated with harvesting procedures. A synthetic, bone graft substitute that can match the clinical performance of autogenous bone could alleviate these deficiencies. Therefore, a polymeric bone substitute was developed that consists of a four-armed star polymer of poly(dioxanone-co-glycolide) endcapped at each termini with a biocompatible lysine-based diisocyanate crosslinker. The polymer can be mixed with inorganic fillers such as either hydroxyapatite or tricalcium phosphate to fotm either injectable or moldable putty. The addition of a catalyst (for example, diethylaminoethanol and water) to the polymer produces a crosslinking reaction causing the combination to harden. This reaction is nontoxic, normo-thermic and can be performed in situ. During the course of the polymerization, carbon dioxide is liberated, producing an
interconnected porous network within the implant, suitable for bone ingrowth. This paper will describe a preliminary biocompatibility assay of the bone substitute.
INTRODUCTION
An effic,acious, resorbable, synthetic bone graft substitute should promote bone ingrowth and resorb in a predictable manner in register with new bone formation (for a recent review see 2). The
Address for correspondence and reprints: E. A. Gruskin, United States Surgical Corporation, 195 McDermott Ave., North Haven, CT 06473.
0 1996 by Elsevier Science Inc. All rights reserved.
101s
8756-3282/96/$15.00 PI1 S8756-3282(96)00130-5
102s
S. Bennett et al. Degradable polymeric
bone substitute
Bone Vol. 19, No. I, Supplement July 1996:101S-107s
capability for bone ingrowth into a porous implant is underscored by the bone-implant interface. This interface must prevent soft tissue intrusion that can inhibit bone growth. A rigid implant may not be suitably contoured and mortised into an irregular recipient bed. Therefore a moldable, or injectable material that hardens in situ and adapts to interface contours will have significant clinical utility. For example, Constanz and coworkers developed a liquid mixture of inorganic minerals that crystallizes into dahllite after introduction into fracture sites. The resulting micro-porous implant erodes from the surface and is replaced by bone in a coupled process (1). An alternative to a nonporous, erodable implant is to provide a porous scaffold for bone ingrowth. For example, Mikos and coworkers described a system that consists of a mineral component and unsaturated, linear polyesters that are polymerized in situ by a free radical-based reaction. The material includes 250-425 pm sodium chloride particles that dissolve, leaving behind a porous, polymer and mineral composite (6). The bone graft substitute we developed can be introduced as a putty to conform to the geometry of the bone defect. To create pores we used a non-toxic moiety that causes the release of carbon dioxide during the polymerization reaction. In addition to generating interconnected pores, the carbon dioxide causes expansion of the implant The result is a porous bone graft substitute with a seamless graftbone interface that allows bone ingrowth and prevents soft tissue prolapse.
Our novel polymer system that hardens in situ into a foam-like porous implant consists of a hydroxyl-terminated biodegradable polymer end capped with a reactive lysine-based diisocyanate moiety. To achieve a three dimensional crosslinked network, the polymeric base unit must have more than two reactive end-groups. Therefore, a “star polymer” ofp-dioxanone and glycolate was developed with four crosslinkable ends. Since the polymer was designed to be a liquid at room temperature, there is no need for a separate solvent to dissolve the reactants. Linked to each terminal hydroxyl group is a reactive crosslinker, similar to diisocyanate, but designed to be nontoxic. The result is a reactive “star polymer” primed to form an intricate three dimensional network upon polymerization. The polymer can be combined with either hydroxyapatite or tricalcium phosphate to form lactomer@ diisocyanate putty (LDIP). Prior to implantation, a catalyst is added to drive the crosslinking reaction. Since the diisocyanate group is used to synthesize polyurethane and polyurea, its crosslinking reactions have been thoroughly characterized (4). In this case, polymerization is catalyzed by water and a tertiary amine and carbon dioxide is liberated. Carbon dioxide release causes a foaming reaction that expands LDIP to fill voids in the bone defect. The reaction is complete in about ten minutes and the porous implant is retained at the recipient site through mechanical interlocking. In addition, the porous network has an architecture similar to bone, further enhancing the biomimetic effect of the bone substitute (FIG. 2).
In this report we describe the synthesis of LDIP and show an initial biocompatibility study. LDIP was polymerized in molds and implanted in the pectoralis muscles in rats. The implants were
Bone Vol. 19, No. 1, hpplement July 1996:101S-107s
S. Bennett et al. Degradable polymeric bone substitute
evaluated clinically and histologically at days 1,3,7, 14,21, and 42 for host tissue responses. Over the course of the study, the tissue responses to the implanted polymer were without unusual sequelae.
MATERIALS
& METHODS
Polymer Svnthesis The star polymer backbone of poly(dioxanone-co-glycolide) (PDO/G *) was prepared using 48.0 g pentaerythritol (Aldrich, Milwaukee, WI), 164.9 gp-dioxanone (United States Surgical Corp., Norwalk, CT), 24.1 g glycolide (United States Surgical Corp., Norwalk, CT), and 0.6 g stannous octoate (Brand NuLabs, Meriden, CT). The reactants were combined under nitrogen, heated to 90 oC for 60 hrs with stirring and then subjected to vacuum
. .. Biocompaublhty Sh.Q LDIP w,as prepared with either hydroxyapatite or p-tricalcium phosphate (10 pm particle size, Himed Inc, Gld Bethpage, NY). Standardized implants were formed by polymerizing LDIP in 3 mm diameter by !j mm cylindrical Teflon molds and the resulting porous rods were measured to confii uniformity. All implants were inserted surgically in the pectoralis muscles of rats following standard operating procedures (3). The rats were euthanized at days 1,3,7, 14,21 and 42 and the implant sites were examined clinically and histologically.
103s
104.9
S. Bennett et al. Degradable polymeric
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
bone substitute
II
I
0
+
KCl,
CbC
*
Iv
m
oc
TEA, 40 brs, RT
0
oc\
7
OCN V
FIG. 1: Schematic representation of LDI synthesis.
RESULTS
& DISCUSSION
Clinical examination did not reveal any signs of host incompatibility. Histologically, a transient inflammatory reaction occurred until day 3, at which time a minimal number of foreign body giant cells were observed. However, after the initial inflammatory reaction, the local response revealed a
Bone Vol. 19, No. 1, Supplement July 1996:1OlS-107s
S. Bennett et al. Degradable polymeric bone substitute
rich fibro-vascular sheath, without evidence of a sustained inflammatory reaction. The polymer implants were well integrated with the host tissue. In addition, there were numerous vascular buds that grew into several implants (FIG. 3 and 4). Importantly, concurrent with this process was a fibrovascular penetration of the implant, thus indicating degradation byproducts were not posing a toxic challenge.
FIG. 2: Scanning electron microscope ev&&n
of the interface between
The
bone-implant interface crosses vertically in the center of the image.
CONCLUSIONS
Preliminary biccompatibility data appear to indicate that LDIP does not elicit an adverse tissue response following an intramuscular implantation. The polymer surface appears to be an acceptable substrate for cell attachment. Any byproducts from the polymerization reaction do not cause significant toxicity and the degradation products are well tolerated. Additional experiments are planned for o’rthopic sites to assess the osteoconductive potential of this class of polymers for bone repair.
105s
106s
S. Bennett et al. Degradable polymeric
*y-to . FI Panel
* 1 i
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
bone substitute
.
. .
.
ralis muscle&.
A; day 3; Panel B; day7; PanelC; day 14; Panel D; day21; PanelE; day42.
A
g
. .
.
mtncalciump&e
in rat m
muscles: Panel A; day 3; Panel B; day 7; Panel C; day 14; Panel D; day 21; Panel E; day 42.
Bone Vol. 19, No. 1, Supplement July 1996:101S-107s
S. Bennett et al. Degradable polymeric bone substitute
REFERENCES
1) Constanz BR, Ison IC, Fulmer MT, Poser RD, Smith ST, VanWagoner M, Ross J, Goldstein SA, Jupiter JB, and Rosenthal DI. Skeletal Repair by in situ Formation of the Mineral Phase of Bone. Science 267: 1796- 1799; 1995. 2) Gazdag AR, Lane JM, Glaser D, and Forster RA. Alternatives to Autogenous Bone Graft: Efficacy and Indications. J. of the Amer. Acad. of Orthopaedic Surgeons 3: 1-8; 1995. 3) Katz RW, Hollinger JO and Reddi A.H. The funcional equivalence of demineralized bone and tooth matrices in ectopic bone induction. J Biomed Mater Res 27:239-245; 1993. 4) March J. Advanced Organic Chemistry. John Wiley & Sons, New York Chichester Brisbane Toronto Singapore, ~786; 1985. 5) Storey RF, Wiggins JS, Mauritz KA and Puckett AD. Bioadsorbable composites. II: Nontoxic, L-lysine-based poly(ester-urathane) matrix composites. Polymer Composites 14 :17-25; 1993. 6) Yaszemski MJ, Payne RG, Hayes WC, Langer RS, Aufdemorte TB and Mikos AG. The Ingrowth of New Bone Tissue and Initial Mechanical Properties of a Degrading Polymeric Composite Scaffold. Tissue Engineering I:41-52; 1995.
107s