JBJS | Delivery Systems for BMPs: Factors Contributing to Protein Retention at an Application Site

The Journal of Bone & Joint Surgery, Volume 83, Issue 1 suppl 2

Delivery Systems for the BMPs | April 01, 2001

Delivery Systems for BMPs: Factors Contributing to Protein Retention at an Application Site

Hasan Uludag, PhD; Tiejun Gao, PhD; Thomas J. Porter, PhD; Wolfgang Friess, PhD; John M. Wozney, PhD

From Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada; Biopharmaceutical Characterization and Analysis Group and Bone Biology and Applications Group, Genetics Institute, Inc., Andover, Massachusetts, U.S.A.; and Department of Pharmaceutical Technology, University of Erlangen, Erlangen, Germany
Hasan Uludag, PhD
Tiejun Gao, PhD
Department of Biomedical Engineering, Faculty of Medicine and Dentistry, University of Alberta, 1098 EDC Building, Edmonton, Alberta T6G 2V2, Canada. E-mail address for Hasan Uludag: hasan.uludag@ualberta.ca

Thomas J. Porter, PhD
Biopharmaceutical Characterization and Analysis Group

John M. Wozney, PhD
Bone Biology and Applications Group
Genetics Institute, Inc., One Burtt Road, Andover, MA 01810, U.S.A.

Wolfgang Friess, PhD
Department of Pharmaceutical Technology, University of Erlangen, Cauerstrasse 4, Erlangen 91058, Germany

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Genetics Institute, Inc. In addition, one or more of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (Genetics Institute, Inc.). No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

The Journal of Bone & Joint Surgery. 2001; 83:S128-S135

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Abstract

Background: Recombinant human bone morphogenetic proteins (rhBMPs) are being tested in clinical studies for their capacity to elicit bone formation. Biomaterials used in delivery systems also play a critical role in supporting the osteoinductive activity of BMPs, attributable to the controlled presentation of the BMPs to target cells. Despite extensive preclinical studies, the factors contributing to local rhBMP pharmacokinetics remain to be elucidated.

Methods: The rhBMP pharmacokinetics were studied in a rat subcutaneous implant and in an intramuscular injection model. In situ levels of rhBMPs were quantitated with use of ¹²⁵I-labeled tracers. The effects of protein structural features and the nature of the biomaterial implant were explored. Osteoinduction by biomaterial+rhBMP combinations was assessed by a semiquantitative, histology-based bone score.

Results: With the use of rhBMP-2, rhBMP-4, and an N-truncated rhBMP-2, the protein isoelectric point was found critical for the initial retention of rhBMPs in an implant. Osteoinduction studies carried out in parallel indicated that rhBMPs with a higher implant retention elicited more bone formation. In the clinically used collagen+rhBMP-2 device, collagen crosslinking and sterilization were most influential in rhBMP-2 retention. To increase retention at an application site, thermoreversible polymers were engineered and shown to enhance local rhBMP-2 retention, especially by injectable delivery.

Conclusions: Two critical components of an osteoinductive device—namely, the biomaterial and the rhBMP—were shown to influence local protein pharmacokinetics and osteoinductive activity of the device. Designer biomaterials can provide an additional mechanism to modulate local protein pharmacokinetics.

Clinical Relevance: These studies form the foundation of next-generation osteoinductive devices with improved potency at sites of desired bone regeneration and reduced side effects at other sites.

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Introduction

Introduction | Materials and Methods | Results and Discussion | References

Bone morphogenetic proteins (BMPs) induce differentiation of uncommitted mesenchymal stem cells, and possibly other target cells, into mineral-depositing osteoblasts^1,2. Although BMPs possess activities beyond osteoinduction², the primary interest in the proteins arises from their potential use in clinical bone regeneration. The early work on BMPs ultimately led to cloning of BMP-2 and BMP-7 (also known as osteogenic protein-1)^3,4, which are under clinical investigation for local bone induction^5,6. The proteins are being investigated as part of a device in combination with a biomaterial carrier (delivery system). In preclinical studies, numerous carriers were shown to be compatible with the osteoinductive activity of recombinant human (rh) BMPs⁷; however, only collagen-based carriers are being used in a clinical setting. An absorbable collagen sponge (ACS), reconstituted from bovine tendon, and a collagen-based matrix, derived from demineralized/guanidine-extracted bovine bone, have been used for the delivery of rhBMP-2 and rhBMP-7, respectively. These choices have been based on a series of preclinical studies, which indicated a pharmacological stimulation of local bone induction by both proteins. An added advantage of collagen is that it is a natural component of bone whose degradation and degradation products can be mediated by physiological means.

When a biomaterial is used for the delivery of a BMP, one would expect the biomaterial to support and contribute to the local bone-induction activity of the BMP. The importance of biomaterials was recognized early when it was observed that (i) the induced bone typically occurred within or in close proximity to the biomaterial, (ii) the dose needed for an efficacious bone induction was significantly reduced, and (iii) the reproducibility of the bone induction cascade was improved when BMPs were implanted with biomaterials.

The mechanism(s) by which a biomaterial influences osteoinductive activity is an area of active investigation. One mechanism is the direct interaction of a biomaterial with the target cells on which BMPs act. A cell-compatible biomaterial helps to support cell proliferation and, by providing a suitable attachment substrate, can directly influence cellular differentiation into an osteogenic phenotype⁸. A second mechanism by which a biomaterial acts is by binding to BMPs and presenting them to the target cells in a "bound" form. The local concentration of the BMPs may be elevated because it is sequestered in a carrier. A biomaterial might also potentiate the activity of BMPs by binding and presenting the proteins to cell receptors directly (i.e., an enhanced activity compared with a freely diffusable protein)⁹. In contrast to bound BMP, slowly released BMP might be critical for the activity by providing a physiological concentration of free BMP available in the vicinity of an implant for a prolonged time. The released BMP might also attract target cells to a desired site of bone induction by chemotaxis¹⁰. A technical challenge—namely, to differentiate between freely diffusable compared with bound protein in vivo—has not made it feasible to elucidate such mechanisms.

The determination of the exact mechanism of BMP action requires knowledge of in situ protein pharmacokinetics. Toward this goal, we initiated a series of studies with rhBMP-2^11,16 with the ultimate aim of (i) probing factors influencing rhBMP pharmacokinetics and (ii) exploring the correlation between the pharmacokinetics and osteoinductive activity. In this paper, we first examine the correlations between the in situ retention of rhBMPs and osteoinduction. Second, we provide a summary of ACS collagen sponge properties that contribute to rhBMP-2 retention. Third, we propose a novel approach for rhBMP-2 retention at an application site. The latter was based on engineered thermoreversible biomaterials, which undergo temperature-dependent phase transition. By manipulating rhBMP pharmacokinetics, we aim to obtain osteoinductive devices with improved osteopotency and minimal bone formation at undesirable sites.

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Fig. 1:: Mean ± SD percent retention of recombinant human bone morphogenetic protein-2 (rhBMP-2) compared with plasmin-cleaved rhBMP-2 (A) and rhBMP-2 compared with rhBMP-4 (B) in implants. The difference between the proteins manifested itself in the first explantation time (3 hours), whereas proteins with a lower isoelectric point (pI) had lower rates of retention. (Fig. 1-A was reprinted, with permission, from Winn S, Uludag H, Hollinger JO. Carrier systems for bone morphogenetic proteins. Clin Orthop. 1999;367S:S95-S106. Copyright © 1999, Lippincott. Fig. 1-B was reprinted, with permission, from Uludag H, D’Augusta D, Golden J, Li J, Timony G, Reidel R, Wozney JM. Implantation of recombinant human bone morphogenetic proteins with biomaterials. J Biomed Mat Res. 2000;50:227-38. Copyright © 2000, John Wiley & Sons.)

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Fig. 2:: Percent trichloroacetic acid (TCA)-precipitable counts (mean ± SD). The recombinant human bone morphogenetic protein-2 (rhBMP-2) and plasmin-cleaved rhBMP-2 solutions were kept in vitro at 4°C for 7 and 13 days and TCA-precipitated either from the solution or after applying the solution to sponges. In parallel, radioactive counts in 7-day and 13-day subcutaneous implants were homogenized and TCA-precipitated. For day-13 explants, the precipitated counts were further homogenized with 4 M guanidine and precipitated. There were no significant TCA-soluble counts after the first precipitation (not shown).

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Fig. 3:: Osteoinductive activity of recombinant human bone morphogenetic protein-2 (rhBMP-2) compared with plasmin-cleaved rhBMP-2 (A) and rhBMP-2 compared with rhBMP-4 (B). Bone formation was assessed after 13 (A) or 14 days (B) of implantation and expressed as mean SD bone score for six implants at each dose. More potent rhBMP (rhBMP-2 in both A and B) was evident by bone induction at a lower dose.

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Fig. 4:: A histological section of mineralized tissues induced by plasmin-cleaved recombinant human bone morphogenetic protein-2.

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Fig. 5:: Mean SD percent retention of recombinant human bone morphogenetic protein (rhBMP-2) in different collagen sponges. Untreated sponges (uncrosslinked/unsterile) had the highest initial retention but also exhibited the fastest rate of rhBMP-2 loss. Fully processed sponges had the highest rate of rhBMP-2 retention after 3 days. (Reprinted, with permission, from Winn SR, Uludag H, Hollinger JO. Sustained release emphasizing recombinant human bone morphogenetic protein-2. Adv Drug Del Rev. 1998;31:303-18. Copyright © 1998, Elsevier.)

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Fig. 6-A:: Fig. 6-A Monomers used for thermoreversible polymers.

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Fig. 6-B:: Fig. 6-B Optical density of polymer solutions as a function of temperature. The lower critical solution temperature (LCST) was 26.7, 19.4, and 18.5°C for N-isopropylacrylamide (NiPAM) (circle), NiPAM/ethylmethacrylate (EMA) copolymer (square), and NiPAM/N-acryloxysuccinimide copolymer (NASI) (diamond), respectively.

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Fig. 6-C:: Fig. 6-C Conjugation of recombinant human bone morphogenetic protein-2 (rhBMP-2) to NiPAM/NASI polymers. Lane 1 was MW standards; lane 2, control rhBMP-2 (no polymer); and lanes 3, 4, and 5 contained rhBMP-2:polymer ratios of 1:128, 1:80, and 1:40, respectively.

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Fig. 7:: Pharmacokinetics of recombinant human bone morphogenetic protein-2 (rhBMP-2)+thermoreversible biomaterials in subcutaneous implantation (A) and intramuscular injection (B) models. The biomaterials were incubated with rhBMP-2 for 3 hours at 4°C in 0.1 M phosphate buffer (pH 7.4). The solutions were then diluted with glycine buffer containing the same biomaterials to obtain a biomaterial concentration of 28.7 mg/ml. The rhBMP-2 concentration was 30 g/ml. Note that in the implant model, the N-isopropylacrylamide (NiPAM)/N-acryloxysuccinimide copolymer (NASI) was most effective in retaining rhBMP-2, whereas in the injection model NiPAM/NASI and NiPAM/ethylmethacrylate copolymer (EMA) were equally effective. The biomaterials were more effective in the injection model due to rapid loss of free rhBMP-2 in this model.

Materials and Methods

Introduction | Materials and Methods | Results and Discussion | References

Materials

Helistat® sponges were from Integra Life Sciences (Plainsboro, New Jersey). The sponges were prepared with the use of Type I collagen, which was treated with formaldehyde for crosslinking and with ethylene oxide for sterilization¹³. For a particular study, partially treated sponges were removed during these procedures, before the formaldehyde treatment to give uncrosslinked/unsterile sponges, and before the ethylene oxide treatment to give crosslinked/unsterile sponges. Fully processed (crosslinked/sterile) sponges from this lot were used as the control for that particular study. Iodo-Gen® and ¹²⁵I were from Pierce (Rockford, Illinois) and NEN (Boston, Massachusetts), respectively. Male Long-Evans rats, 4-5 weeks old, were obtained from Charles River Labs (Portage, Michigan) for subcutaneous implantation. Female Sprague-Dawley rats, 4-6 weeks old, were obtained from Biosciences (Edmonton, Alberta, Canada) for conjugate implantation and injection studies.

rhBMPs

The rhBMP-2 and rhBMP-4 were produced in CHO cells and dissolved in 0.5 M arginine, 10 mM histidine, pH 6.5, or 2.5% glycine, 0.5% sucrose, 0.01% Tween-80, 5 mM glutamic acid, 5 mM NaCl, pH 4.5, buffers. Plasmin-cleaved rhBMP-2 was prepared by mixing rhBMP-2 with plasmin at 1:10 molar ratio. The mixture was incubated for 24 hours, and the protein was purified by cation exchange chromatography and desalted by reverse-phase high-pressure liquid chromatography (RP-HPLC¹¹). As control, native rhBMP-2 was desalted by RP-HPLC, lyophilized, and reconstituted with glycine buffer. The N-terminal amino acid sequence was determined with an Edman sequencer. The isoelectric point (pI) of the proteins was determined by isoelectric focusing (IEF) gels¹².

Proteins used in pharmacokinetics studies were labeled with ¹²⁵I by Iodo-Gen¹¹. The labeled protein was then added to a cold protein solution to give a final ¹²³I-labelled:unlabeled protein, ratio of >1:100, which corresponds to the ratio of unlabelled rhBMP to rhBMP used in the iodination process. Trichloroacetic acid (TCA) precipitation of the final solutions routinely showed >95% protein-bound counts.

Synthesis and Characterization of Thermoreversible Polymers

The preparation of N-isopropylacrylamide (NiPAM)-based biomaterials was reported previously¹⁶. The polymer compositions were determined by ¹H NMR. The lower critical solution temperatures (LCST; temperature at which soluble insoluble transition occurs) of polymers were determined by spectroscopy¹⁶. The conjugation reaction between rhBMP-2 and polymers was investigated by mixing a polymer solution (in phosphate buffer, pH 7.4) with a rhBMP-2 solution (in MES buffer, pH 4.5; buffer exchanged from the glycine buffer) at 4°C and analyzing the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)¹⁶.

Pharmacokinetics Assessment

The BMP pharmacokinetics were investigated in a subcutaneous implantation¹¹ or an intramuscular injection model¹⁶.

Subcutaneous implantation: All animals received two subcutaneous implants in the ventral thoracic region through a 5-mm incision¹¹. The implants consisted of 14 14 mm sponges (thickness: 3.5 mm, dry volume: 680 l) cut from 3 4-inch Helistat®. The sponges were soaked with 2 ml rhBMP solution (29%) in petri dishes and were allowed to incubate for 10-30 minutes before implantation. In studies designed to compare the pharmacokinetics of structurally related rhBMPs, protein concentrations were either 0.02 mg/ml (rhBMP-2 compared with plasmin-cleaved rhBMP-2) or 0.05 mg/ml (rhBMP-2 compared with rhBMP-4). In the study designed to investigate sponge processing parameters, the concentration of rhBMP-2 solution was 1.5 mg/ml (¹²⁵I-labelled:unlabelled protein ratio of 1:4000). In the conjugate implant study, rhBMP-2 solution containing ¹²⁵I-rhBMP-2 (2.4 mg/ml) was incubated with a polymer solution (30 mg/ml in 0.1 M phosphate buffer) for 3 hours at 4°C. The mixture was then diluted to glycine buffer to give a final rhBMP-2:polymer concentration of 30 g/ml:28.7 mg/ml (1:950 rhBMP-2:polymer ratio). From a pool of 12 rats in each group, two rats were sacrificed by CO2 inhalation at the desired time points.

Intramuscular injection: The rhBMP-2 solutions containing a biomaterial (30 g/ml rhBMP-2 and 28.7 mg/ml biomaterial) were directly injected into the gluteus maximus muscle in hindlimbs (two per animal). The rhBMP-2 and the biomaterial were typically mixed for 3 hours at 4°C in 0.1 M phosphate buffer and then diluted with the glycine buffer containing the same biomaterial. One hundred microliters of solution was injected in each site with use of an insulin syringe. At sacrifice, the gluteus maximus was harvested and counted as a whole.

Pharmacokinetics Analysis

Radioactivity associated with explants was measured directly with a g-counter and used as a measure of rhBMP-2. Implants containing native and plasmin-cleaved rhBMP-2 were further processed to determine if the measured counts were protein-bound. Explants from days 7 and 13 were homogenized in phosphate buffered saline (PBS) and precipitated with 10% TCA/1% albumin. The 13-day explants were additionally precipitated in the presence of 4 M guanidine after PBS homogenization. The supernatant was separated from the pellet, and the percentage of precipitable counts was determined by measuring the counts in each fraction. The mean ± SD of four implants at each time point (two per animal) was calculated. All counts were corrected for decay (¹²⁵I half-life of 60 days) and are shown as time = 0 (designated as the implant time) counts. The percentage implanted dose versus time curves were generated by dividing the explant counts by the counts originally implanted. Noncompartmental analysis¹¹ was carried out to calculate a mean residence time (MRT), based on partial and total areas under the curve (AUC), and partial and total areas under the moment curve (AUMC)¹¹. A difference of >± 20% between two parameters was considered significant¹¹. This statistical measure is used to investigate bioequivalence of pharmaceutical formulations.

Osteoinductive Activity

The osteoinductive activity of rhBMP+biomaterial devices was investigated in the subcutaneous implant model. The implant preparation and implantation were as described in the pharmacokinetics section. A range of rhBMP doses (see figures) was implanted, and the amount of de novo bone was assessed after 13 (rhBMP-2 compared with plasmin-cleaved rhBMP-2) or 14 days (rhBMP-2 compared with rhBMP-4). Each group had three rats; this resulted in six implants per group. The explants were fixed with 10% formaldehyde, cut in half, embedded in JB-4 resin, sectioned (5 m), and stained with von Kossa, followed by toluidine blue, and then coverslipped. Histological scoring of newly formed bone was performed under the microscope according to the semiquantitative scoring system¹²: implants containing 0%, <10%, 10-20%, 20-40%, 40-60%, 60-80%, and 80-100% of newly formed bone were assigned a bone score of 0, 0.5, 1, 2, 3, 4, and 5, respectively.

Results and Discussion

Introduction | Materials and Methods | Results and Discussion | References

Pharmacokinetics and Osteoinductive Activity of Structurally Related rhBMPs

The rhBMPs utilized for this study were rhBMP-2, rhBMP-4, and plasmin-cleaved rhBMP-2. The former two are closely related in amino acid sequence and exhibit a similar activity in vitro¹¹. The rhBMP-2 contains a single plasmin cleavage site (per subunit) at its N-terminus. This site can potentially be cleaved by endogenous plasmin in situ, which may influence interaction of the protein with extracellular matrix proteins, heparin, and other glucosaminoglycans, as well as cells responsive to BMP signaling¹⁷. Plasminogen activation to yield active plasmin typically occurs in wound-healing sites¹⁸. The plasmin cleavage resulted in a truncated rhBMP-2 whose N-terminus was reduced from TFGHDGKGHPLHKREKRQAKHKQRKRLKSSC... (T273 isoform) to RLKSSC... (R391 isoform), resulting in a pI shift from ~9.0 to 6.5 (N-Terminal amino acid sequence; single letter code for amino acids)¹². The SDS-PAGE analysis indicated no gross changes in molecular weight (only a 1-3 kD shift), consistent with cleavage only at the N-terminus. The active site of BMPs is believed to be within the cystine knot¹⁹, an area unaffected by the cleavage. The rhBMP-4 had a pI of 7.0-8.5 with a number of well resolved species by IEF. However, amino acid sequencing indicated only two isoforms differing in only a single amino acid¹², which cannot account for the observed heterogeneity on IEF gels. Neuroaminadase treatment of rhBMP-4 reduced the heterogeneity, indicating that sialic acid residues are primarily responsible for the lower pI.

The implant concentrations of plasmin-cleaved rhBMP-2 and rhBMP-4 were significantly less than those of the rhBMP-2 throughout the study period (Fig. 1). The differences among the proteins manifested themselves within hours of implantation. The pharmacokinetics analysis yielded a MRT (see Materials and Methods section), which represents the time required for 63% of the dose to be eliminated of 4.3 days for rhBMP-2 compared with 4.2 days for plasmin-cleaved rhBMP-2 and 3.8 days for rhBMP-2 compared with 3.2 days for rhBMP-4. The MRT calculations provided an average rate of protein loss over the study period, where the difference in the initial clearance became negligible. The pAUC, a measure of average dose in an implant, was in line with local retention profiles: 220.9 for rhBMP-2 compared with 47.7 for plasmin-cleaved rhBMP-2, and 154.4 for rhBMP-2 compared with 23.0 for rhBMP-4. Taken together, these results indicated that rhBMPs with lower pI were retained to a lesser extent at an implant site. Several mechanisms might contribute to this observation: (i) a reduced interaction between the rhBMPs in the collagen sponge at lower pI; (ii) a higher solubility in the physiological milieu for proteins with lower pI, resulting in higher mass transport from the implant; (iii) a reduced interaction between the low pI rhBMPs and biological surfaces or proteins in biological fluids; or (iv) any combination of these reasons. The present study did not provide any evidence to distinguish among these possibilities. Elucidation of the rhBMP clearance mechanism(s) might provide novel ways of achieving protein retention at implant sites.

A critical issue with the use of radioactive tracer techniques is the stability of the protein label. The high protein background in biological fluids makes it difficult to follow the biodisposition of an exogenous protein and, in the case of BMPs, the local bone formation makes it even more difficult to extract and quantitate BMP concentrations in newly induced tissues. Alternatively, we investigated whether the measured counts were protein-bound or in free form. Day-7 explants were homogenized and precipitated once, whereas Day-13 explants were homogenized and precipitated three times (Fig. 2). As controls, rhBMP-2 and plasmin-cleaved rhBMP-2 solutions used in implantation were kept in vitro at 4°C and the percent TCA-precipitable fractions were determined by precipitation of solutions or after applying the solutions to collagen sponges. Homogenization with a high-speed blender did not affect the label stability (not shown). Solutions kept in vitro were ~90% and 85% TCA-precipitable after 7 and 13 days. The TCA-precipitable fractions did not change after rhBMP-2 application to collagen sponges. Similarly, the radioactive counts associated with the explants on days 7 and 13 were >93% TCA precipitable. Repeated precipitation of homogenized explants in the presence of guanidine did not give significant TCA-soluble counts (not shown). Taken together, these results indicate that the radioactive counts retained in implants were indeed protein-bound.

The osteoinductive activity of the rhBMPs as a function of implant dose is shown in Figure 3. A dose-dependent increase in osteoinductive activity was evident for all rhBMPs. However, the rhBMPs whose retention was significantly reduced as a result of initial burst release were also less osteopotent; i.e., the threshold dose required for osteoinduction was higher (plasmin-cleaved rhBMP-2 in Fig. 3-A and rhBMP-4 in Fig. 3-B), and the dose required for a full osteoinductive activity (bone score >4.0) was higher (rhBMP-4 in Fig. 3-B). No apparent differences were evident in the qualitative nature of the de novo induced bone by different rhBMPs. An osteoblast population actively depositing unmineralized extracellular matrix, hypertrophied chondrocytes, and hematopoietic cells was present in implants exhibiting clear osteoinduction (Fig. 4). The distribution of the newly deposited mineral, as revealed by the dark Von Kossa stained regions, also did not appear to be dependent on the nature of rhBMP. Taken together, rhBMPs whose retention at an implant site was enhanced were also more osteoinductive.

Collagen Sponge Variable Influencing Local rhBMP-2 Pharmacokinetics

The factors contributing to protein retention in an rhBMP-2+ACS were then investigated to develop a better understanding of device parameters influencing osteoinduction. Among the factors investigated were the rhBMP-2 concentration used for implantation and ACS processing parameters. Between 2 mg/ml (clinical dose range) and 0.08 (small animal dose range), the protein retention was independent on the concentration¹¹. If ACS binding is the primary mechanism of retention, this indicated that the binding capability of the sponge was not saturated in this range. Otherwise, a faster clearance of rhBMP-2 at higher concentrations was expected.

ACS preparation parameters, on the other hand, were found to influence the rhBMP-2 retention. Both formaldehyde crosslinking and ethylene oxide sterilization were previously shown to influence the physicochemical properties of sponges^13,14. A pharmacokinetics study was carried out with three types of sponges: uncrosslinked/nonsterile (untreated), crosslinked/nonsterile (formaldehyde treated), and crosslinked/sterile (formaldehyde and ethylene oxide treated). The results indicated significant variations in rhBMP-2 pharmacokinetics (Fig. 5). Untreated sponges retained the highest fraction of the implanted dose (87.1 ± 11.3%) after 3 hours, whereas formaldehyde and formaldehyde/ethylene oxide treated sponges retained less. The initial protein loss, however, was highest for the untreated sponges, which resulted in a relatively small MRT of 2.52 days compared with crosslinked/nonsterile and crosslinked/sterile sponges (2.97 and 3.37 day, respectively). The highest pAUC was seen with uncrosslinked/nonsterile sponges, due to the highest initial recovery, followed by crosslinked/sterile and crosslinked/nonsterile sponges (345.5, 195.6, and 269.7, respectively). The high initial retention by the unprocessed sponges (the highest among the biomaterials investigated in our studies) was indicative of better rhBMP-2 binding by the native collagen itself. This was supported by in vitro binding studies in which ~98% of applied dose was typically bound¹³. A significant drawback was the rapid rhBMP-2 loss (the fastest observed among collagen carriers), which was indicative of fast collagen degradation in vivo. Alternative crosslinking techniques might be desirable in which the rhBMP-2 binding motifs are not compromised while in vivo collagen resiliency is enhanced. The fully processed sponges gave the highest local rhBMP-2 concentration after 14 days. Although some variations in physicochemical properties of fully processed sponges were observed, such variations did not lead to differences in rhBMP-2 retention¹⁵.

Control of?in Situ Retention of rhBMP-2 by Thermoreversible Biomaterials

Our studies indicated that rhBMP retention in osteoinductive devices is dependent on ACS properties and protein structural features. To explore additional mechanisms of retention, synthetic water-soluble biomaterials were designed that can be used as a supplement in aqueous rhBMP formulations. Synthetic polymers were chosen to precisely tailor the physicochemical properties important for protein retention. Temperature-sensitive polymers were suitable for this purpose, since the polymers can be formulated (i.e., remain in solution) in aqueous buffers at a low temperature but become insoluble when delivered to the physiological milieu. Temperature-dependent solubility avoids the use of additional exogenous agents to induce a phase change. By facilitating the interaction of insolubilized biomaterial with a co-delivered rhBMP, it was our intention to enhance the rhBMP retention at an application site.

To demonstrate the feasibility of this approach, we employed NiPAM-based polymers that are cell compatible in vitro²⁰. The LCST was considered a critical property for the polymers because it was expected to determine the in vivo resiliency of the biomaterials. Whereas charged and hydrophilic residues increase the LCST, hydrophobic residues, such as alkylmethacrylates, decrease the LCST¹⁶. A library of NiPAM-based polymers were synthesized with a range of properties¹⁶and three were chosen for in vivo studies (Fig. 6-A): a NiPAM homopolymer, a NiPAM/ethylmethacrylate copolymer (NiPAM/EMA: 73.7/26.3%), and a NiPAM/N-acryloxysuccinimide copolymer (NiPAM/NASI: 92.8/7.2%). The EMA was utilized to lower the LCST, whereas NASI was utilized to introduce protein-NH2 reactive groups into polymers. The LCST of NiPAM homopolymer was 26.7°C, whereas the LCSTs of NiPAM/EMA and NiPAM/NASI were 19.4 and 18.5°C (Fig. 6-B), respectively. As demonstrated by SDS-PAGE analysis, NiPAM/NASI reacted with rhBMP-2 as a function of incubation time but no reaction was seen with NiPAM and NiPAM/EMA (not shown). The conjugation efficiency was dependent on the relative concentration of NiPAM/NASI to rhBMP-2. Some conjugation was seen at a polymer:rhBMP-2 ratio of 40:1, but complete conjugation was obtained at 80:1 and 128:1 ratios after a 3-hour reaction (Fig. 6-C). Therefore, in vivo PK studies were carried out at a rhBMP-2:polymer ratio of >130:1 where all rhBMP-2 were polymer conjugated.

At a polymer concentration of 28.7 mg/ml, NiPAM/NASI conjugated rhBMP-2 had a higher rate of retention in implants after 5 days but the other polymers were not effective in enhancing retention (Fig. 7-A). In contrast, when the formulations were delivered intramuscularly by injection, significant differences among the study groups were evident (Fig. 7-B). Whereas NiPAM and control rhBMP-2 produced a similarly low rate of retention, NiPAM/EMA and NiPAM/NASI gave an ~2-fold increased rhBMP-2 retention on day 1 and 17-21-fold and 218-242-fold higher rates of retention on days 5 and 9, respectively. These results indicate that the designed polymers were more effective in an injectable format. This was not surprising, since injecting free rhBMP-2 in conventional formulations was expected to give rapid protein loss. However, the presence of ACS also appeared to reduce the effectiveness of the polymers to sequester the rhBMP-2. Both physical entrapment (NiPAM/EMA) and chemical conjugation (NIPAM/NASI) mechanisms were effective in enhancing retention. By relying on thermoreversible biomaterials for rhBMP-2 retention, it might be feasible to engineer a scaffold without affecting its properties that are responsible for rhBMP-2 retention. A critical issue, the compatibility of thermoreversible biomaterials with rhBMP-2 induced bone induction, needs to be determined and such studies are currently underway?²¹. Because the designed biomaterials were especially effective in an injectable format, they might pave the way for injectable delivery of rhBMPs in a clinical setting, possibly eliminating the need for invasive surgical implantation.

rhBMP Pharmacokinetics at Other Anatomical Sites

In investigating rhBMP-2 pharmacokinetics, we chose to utilize rat ectopic systems. A critical issue is the applicability of the results to other preclinical models and the clinical situations. Other animal models typically use larger size implants with different geometry, and implants may be exposed to a different environment. The ACS+rhBMP-2 device was recently implanted in full-thickness articular cartilage defects²² and the pharmacokinetics parameters were determined: t1/2 and MRT were 5.6 and 8 days, respectively. These parameters were in the same range as those obtained from the ectopic model, albeit slightly higher. The higher parameters might be due to microenvironmental effects (relatively less vascularized site of cartilage) or reflect differences in the assessment technique (i.e., retrieval compared with in situ scintigraphic imaging²²). The rhBMP-2 pharmacokinetics were recently reported in a rabbit ulnar osteotomy model (0.5 mm)²³. That study provided similar rhBMP-2 pharmacokinetics in the osteotomy site compared with ectopic sites (retention rates in the first 2-3 days and after 14 days were similar, but the osteotomy model retained slightly higher rhBMP-2 at the intermediate time points). Although more detailed studies remain to be reported, these preliminary data suggest that rhBMP pharmacokinetics from the ectopic sites are comparable with those from other anatomical sites. This makes the ectopic models useful to design osteoinductive devices with improved potency and reduced side effects.

Note: We thank D. D"Augusta, C. Blake, R. Palmer, and N. Kousinioris for animal surgery, J. Golden and J. Li for histology, G. Timony for pharmacokinetics analysis, and X.-D. Fan for polymer synthesis. These studies were carried out at Genetics Institute Inc. and University of Alberta. The studies at the University of Alberta were supported by Genetics Institute and MRC of Canada. T. J. Gao is an Alberta Heritage Foundation for Medical Research (AHFMR) research fellow.

References

Introduction | Materials and Methods | Results and Discussion | References

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