Introduction: Delivery of functionally active molecules to a variety of musculoskeletal tissues has been a major focus of investigation. However, use of biologically active proteins is hindered by a high cost of production and difficulty with long term storage. Peptides are an attractive alternative to active proteins because of their relatively simple structure and greater stability. Both of these traits reduce the cost and complexity of delivering bioactive molecules for tissue engineering. In vivo biopanning studies have revealed bone targeting peptides that potentiate bone regeneration. One of the bone targeting peptides, when delivered to rat bone defects, has been shown to enhance and accelerate bone healing. Local delivery of the peptide in a controlled and sustained manner using appropriate carrier vehicles is important to healthy bone growth. Various polymeric controlled nano/micro sized delivery vehicles are currently under development for tissue engineering applications. Among these, injectable in situ gelling hydrogels have tremendous potential as ideal carrier matrices for a wide range of biomedical and pharmaceutical application such as drug delivery and tissue engineering [1]. The advantages of gelling systems over preformed matrices include introduction into the body in a minimally invasive manner, the ability to conform to complex shapes, and delivery of bioactive molecules or cells to the defect site under mild conditions. For effective clinical application, injectable systems should gel at mild physiological conditions in a short time period. An injectable thermosensitive system based on the biocompatible natural polymer chitosan has been developed as a delivery vehicle for the peptide [2]. In the presence of certain inorganic phosphate salts, such as ammonium hydrogen phosphate, the liquid chitosan solution exhibits thermosensitive behavior and is transformed into a gel at clinically feasible times ranging from 2 to 10 minutes at 37 °C. We hypothesize that this novel thermosetting injectable system will serve as a viable carrier vehicle to deliver osteogenic peptides locally to the defect site to enhance bone repair [3].

Materials and Methods: Biomedical grade chitosan (84.2% and 78.9% block deacetylated) was purchased from BioSyntech, Quebec, Canada. Non-biomedical grade chitosan (85% minimum deacetylated) and Ammonium Hydrogen Phosphate (AHP) were purchased from Sigma-Aldrich, St. Louis, USA. Preparation of thermogel: The thermogelling solution is prepared by thoroughly mixing the appropriate amount of the AHP salt into a liquid solution of chitosan dissolved in 0.5% acetic acid. The viscosity of the chitosan solutions used were 1500 cP. The AHP salt is dissolved in distilled deionized water at a concentration of 6 grams per 10 mL water. The volume added to the chitosan solutions was determined by the amount required to gel the solutions in 10 minutes. The amount of AHP used was 60 µL per mL of 84.2% DDA chitosan solution (~1.6% w/v), 150 µL per mL of 78.9% DDA chitosan solution (~1.4% w/v), and 25 µL per mL of 85% DDA chitosan solution (~2.5% w/v). In vitro release studies: Release of the osteogenic peptide, R1, and bone morphogenetic protein 2 (BMP-2) from the 85% DDA chitosan was studied. The thermogelling solution was mixed thoroughly with the AHP and the desired amount of peptide or protein was added. Three milliliters of the thermogelling solution mixture containing 300 nanomoles of the R1 peptide was submerged in 20 mL of a PBS solution at 37 °C. Samples were collected by completely removing and replacing the released media at specified time intervals and peptides content was analyzed by HPLC. For comparison with a larger polypeptide with osteogenic properties in vivo, the release of 0.3 nanomoles of BMP-2 was studied under the same conditions and with the same volume of gel. The release of BMP-2 was measured using the R&D Systems Quantikine BMP-2 Immunoassay. In vitro degradation studies: Degradation of the 84.2% and 78.9% DDA chitosan thermogels was studied using 1 mL samples. Three samples of each chitosan thermogel were submerged in a PBS solution at 37 °C. Three samples of each gel were also submerged in a lysozyme solution at a concentration of 1 mg lysozyme per 1 mL PBS at 37 °C to better approximate in vivo behavior.

Results and Discussion: In vitro experiments with R1 peptide showed maximum osteogenic activity at a 5 nanomolar concentration [4]. Controlled release of the peptide was attempted in order to release at least this concentration of R1 into the site of a bone defect. To assess the release characteristics from the chitosan thermogel, R1 was added to a thermogelling solution prepared from 85% DDA chitosan. Samples were taken over the course of seven days with six samples taken on the first day to accurately measure a burst release of the peptide. More than 50% of the peptide was released in the first 24 hours and almost 70% was released by day five. The incomplete release of peptide is possibly due to the slow degradation rate of chitosan in PBS. However, the rate of release far exceeded the desired 5 nanomolar per day with almost 5 nanomoles of peptide being release in 20 mL of media even on the fifth day. This release profile demonstrates that although the peptide eluted easily from the polymer matrix, it is possible to prepare a peptide-thermogelling mixture with properties that allow sustained release over several days. The sustained release of high levels of peptide indicates that an even smaller amount of peptide can be used to further reduce the initial concentration of peptide, and therefore the cost of the delivery system. For comparison, the rate of release of recombinant BMP-2 (a larger polypeptide with osteogenic activity) from the same thermogelling system was investigated. Only 4.5% of the BMP-2 was released during the first 24 hours. Effective release of this recombinant BMP-2 protein, which is considerably larger than the R1 peptide, would be entirely controlled by chemical degradation of the chitosan carrier. Degradation of two thermogels made from different biomedical grade chitosans was determined by measuring loss of mass over seven days. In order to avoid impeding the healing of critical size bone defects in a rat animal model, the delivery vehicle must be completely degraded and removed from the site of injury within 5 weeks. Both of the chitosan thermogels that were studied showed sufficient degradation at 37 °C. The thermogel samples that were prepared from 84.2% DDA chitosan lost an average of 57% of their starting mass in just one week in a solution of lysozyme. Samples made from 78.9% DDA chitosan degraded at a much faster rate, losing an average of 87% of their starting mass in the same time period. By contrast, the samples incubated in PBS without lysozyme showed only half of this mass loss due to gel disintegration; the 84.2% DDA chitosan lost an average of only 27% of their mass and the 78.9% DDA chitosan samples an average mass loss of 48%. These results confirm that the thermogels will be largely degraded by lysozyme in vivo and that enzymatic degradation will promote the complete release of peptide or protein. Moreover, changes in the degradation rate properties of the thermogel can be modified by the degree of deacetylation thereby allow for the tailoring of the final release characteristics of biologically active components such as osteogenic peptides from the mixture.

Conclusions: An injectable chitosan thermogel has been shown to possess the desired properties of a carrier for the delivery of a biologically active peptide that promotes the healing of defects in bone. The release rate is sufficient to deliver therapeutic levels of peptide over several days. The rapid degradation rate of the thermogel is sufficient in order not to impede the healing process and, can be tailored according to needs for effective biological activity and clinical uses by modifying the deacetylation of the monomer. Or studies showing that the release of a biologically active peptide from and injectable solution that gels at physiological temperature suggests that further applications makes may be possible for tissue repair and regeneration.

References: 1. Ruel-Gariepy E, Leroux JC. European J Pharma Biopharma 58: 409, 2004. 2. Nair LS & Laurencin CT. US Patent Appl. 60/705, 812 3. Huang D, Beck G, Vedavathi M & Balian G. Orthopaedic Research Society Transactions Vol.31, 152, 2006 4. Vedavathi M, Beck G, Huang D & Balian G Orthopaedic Research Society Transactions Vol.32, 125, 2007