Kimberly A. Hooper

Canine bone response to tyrosine‐derived polycarbonates and poly(L‐lactic acid)

Tyrosine-derived polycarbonates are a new class of degradable polymers developed for orthopedic applications. In this study the long-term (48 week) in vivo degradation kinetics and host bone response to poly(DTE carbonate) and poly(DTH carbonate) were investigated using a canine bone chamber model. Poly(L-lactic acid) (PLA) served as a control material. Two chambers of each test material were retrieved at 6-, 12-, 24-, and 48-week time points. Tyrosine-derived polycarbonates were found to exhibit degradation kinetics comparable to PLA. Each test material lost approximately 50% of its initial molecular weight (Mw) over the 48-week test period. Poly(DTE carbonate) and poly(DTH carbonate) test chambers were characterized by sustained bone ingrowth throughout the 48 weeks. In contrast, bone ingrowth into the PLA chambers peaked at 24 weeks and dropped by half at the 48-week time point. A fibrous tissue layer was found surrounding the PLA implants at all time points. This fibrous tissue layer was notably absent at the interface between bone and the tyrosine-derived polycarbonates. Histologic sections revealed intimate contact between bone and tyrosine-derived polycarbonates. From a degradation-biocompatibility perspective, the tyrosine-derived polycarbonates appear to be comparable, if not superior, to PLA in this canine bone chamber model.

Comparative histological evaluation of new tyrosine‐derived polymers and poly (L‐lactic acid) as a function of polymer degradation

Previous studies demonstrated that poly(DTE carbonate) and poly (DTE adipate), two tyrosine-derived polymers, have suitable properties for use in biomedical applications. This study reports the evaluation of the in vivo tissue response to these polymers in comparison to poly(L-lactic acid) (PLLA). Typically, the biocompatibility of a material is determined through histological evaluations as a function of implantation time in a suitable animal model. However, due to changes that can occur in the tissue response at different stages of the degradation process, a fixed set of time points is not ideal for comparative evaluations of materials having different rates of degradation. Therefore the tissue response elicited by poly(DTE carbonate), poly(DTE adipate), and PLLA was evaluated as a function of molecular weight. This allowed the tissue response to be compared at corresponding stages of degradation. Poly(DTE adipate) consistently elicited the mildest tissue response, as judged by the width and lack of cellularity of the fibrous capsule formed around the implant. The tissue response to poly(DTE carbonate) was mild throughout the 570 day study. However, the response to PLLA fluctuated as a function of the degree of degradation, exhibiting an increase in the intensity of inflammation as the implant began to lose mass. At the completion of the study, tissue ingrowth into the degrading and disintegrating poly(DTE adipate) implant was evident while no comparative ingrowth of tissue was seen for PLLA. The similarity of the in vivo and in vitro degradation rates of each polymer confirmed the absence of enzymatic involvement in the degradation process. A comparison of molecular weight retention, water uptake, and mass loss in vivo with two commonly used in vitro systems [phosphate-buffered saline (PBS) and simulated body fluid (SBF)] demonstrated that for the two tyrosine-derived polymers the in vivo results were equally well simulated in vitro with PBS and SBF. However, for PLLA the in vivo results were better simulated in vitro using PBS.

Comparison of the effect of ethylene oxide and γ-irradiation on selected tyrosine-derived polycarbonates and poly(L-lactic acid)

Tyrosine-derived polycarbonates are a new class of degradable polymers that have possible biomedical applications. In this study, the effect of the two most common sterilization techniques, ethylene oxide and γ-irradiation (0.3, 1.1, 3.9, 6.4, 10.6 Mrad), was evaluated for a family of four structurally related tyrosine-derived polycarbonates and for poly(L-lactic acid) (PLLA). The four polycarbonates were poly(DTE carbonate), poly(DTB carbonate), poly(DTH carbonate), and poly(DTO carbonate) and differed only in the length of the pendent chain. Ethylene oxide exposure had little effect on molecular weight, surface composition, mechanical properties, or degradation rate of all test polymers except for poly(DTO carbonate). Poly(DTO carbonate) was unique since following ethylene oxide exposure it degraded faster than did the nonsterilized control. γ-Irradiated tyrosine-derived polycarbonates retained over 81% of their initial molecular weight when exposed to a clinically relevant dose of 3.9 Mrad and retained still 58% of the initial molecular weight when exposed to the highest test dose of 10.6 Mrad. No changes in surface composition and only slight changes in yield strength and the Youngs modulus were detected for any of the tyrosine-derived polycarbonates following γ-irradiation. In vitro, irradiated films of poly(DTE carbonate), poly(DTB carbonate), and poly(DTH carbonate) degraded at approximately the same rate as did the nonsterilized films regardless of irradiation dose. Only poly(DTO carbonate), irradiated at high doses, degraded faster than did the control. Medical-grade PLLA was tested under identical conditions. Ethylene oxide exposure of PLLA did not affect the molecular weight, surface composition, mechanical properties, or in vitro degradation rate. However, upon irradiation at 10.6 Mrad, PLLA retained only 29% of its initial molecular weight; a dose of 3.9 Mrad resulted in retention of 49% of the initial molecular weight. In correspondence with earlier publications, irradiation of PLLA induced significant losses in the Youngs modulus, % strain at break, and changes in the postirradiation rate of degradation in some specimens. Compared to PLLA, tyrosine-derived polycarbonates are significantly more stable to γ-irradiation and can be sterilized by conventional γ-sterilization techniques.

Characterization of the inflammatory response to biomaterials using a rodent air pouch model.

Using a rodent air pouch, the inflammatory responses to biomaterials with distinct physical properties and chemical compositions were compared. The polymers examined were expanded poly(tetrafluoroethylene) (ePTFE), silicone, low-density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl-tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl-tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. We found that implantation of disks (4.5-4.8 mm) of these materials into rodent air pouches for 2 days had no effect on the number or type of cells recovered relative to sham controls. With each of the materials, macrophages were the predominant cell type identified (60-75%), followed by granulocytes (20-25%) and lymphocytes (10%). Implantation of poly(DTE carbonate), ePTFE, LDPE, or poly(DTBzl carbonate) into the pouches for 2 days caused an increase in release of superoxide anion by the pouch cells. Cells from pouches containing poly(DTE carbonate) also released more hydrogen peroxide and were more phagocytic. In contrast, PLLA and silicone had no effect on the functional activity of cells recovered from the pouches. Prolonging the implantation time of poly(DTE carbonate) or PLLA to 7 days did not alter the number or type of cells isolated from the pouches. However, cells from pouches containing poly(DTE carbonate) for 7 days continued to produce increased quantities of superoxide anion relative to sham control pouch cells. These results suggest that the air pouch model is a highly sensitive method and therefore useful for evaluating the functional responses of inflammatory cells to biomaterials.

Diphenolic Monomers Derived from the Natural Amino Acid α-L-Tyrosine: An Evaluation of Peptide Coupling Techniques

Tlyrosine-derived polycarbonates and polyarylates have recently been recognized as promising biomaterials. In these novel polymers, non-toxic desaminotyrosyl-tyrosine alkyl esters are being used as monomers in place of industrial diphenols such as Bisphenol A. The high cost and limited availability of desaminotyrosyl-tyrosine alkyl esters have prevented the large-scale preparation of these polymers. To address this problem, the following four peptide coupling techniques were explored: dicyclohexylcarbodiimide with 1-hydroxybenzotriazole hydrate (DCC/HOBt), ethyl-3-(3-dimethylamino)propyl carbodiimide hydrochloride salt (EDCI-HCl), N-hydroxysuccinimide (NHS) active ester, and p-nitrophenol (pNP) active ester. Desaminotyrosyl-tyrosine hexyl ester (DTH) was used as a model compound. DCC/HOBt led to a crude product that required column chromatography for purification. The water soluble coupling agent EDCI-HCl made it possible to replace column chromatography by precipitation/extraction in aqueous media. This alleviated possible environmental concerns about the use of organic solvents. Furthermore, EDCI-HC1 did not require the addition of an auxiliary nucleophile such as HOBt in the reaction mixture, The use of the NHS active ester of Dat also produced DTH of sufficient purity, but was less cost effective than EDCI-HCI. The pNP active ester produced YIH which could not be easily purified. Based on these results, the effectiveness of EDCI-HCI was verified by the 100 g synthesis of the ethyl, butyl, hexyl and octyl esters of desaminotrosyl-tyrosine. All of these monomers could be polymerized to high polymers. Overall, the EDCI-HCl mediated coupling of Dat and tyrosine alkyl esters was identified as the best method for the large-scale synthesis of the desaminotyrosyl-tyrosine alkyl esters in a cost efficient and environmentally acceptable manner.

Thermal properties and physical ageing behaviour of tyrosine-derived polycarbonates.

Tyrosine-derived polycarbonates are new carbonate-amide copolymers. These materials have been suggested for use in medical applications, but their thermal properties and their enthalpy relaxation kinetics (physical ageing behaviour) have so far not been evaluated in detail. Since structure-property correlations involving enthalpy relaxation are rarely investigated for biomedical polymers, a series of four tyrosine-derived polycarbonates was used as a model system to study the effect of pendant chain length on the thermal properties and the enthalpy relaxation kinetics. The chemical structure of the test polymers was identical except for the length of their respective pendant chains. This feature facilitated the identification of structure-property correlations. Quantitative differential scanning calorimetry was utilized to determine the thermal properties and to measure enthalpy relaxation kinetics. The glass transition temperature of this family of polymers decreased from 93 to 52 degrees C when the length of the pendant chain was increased from two to eight carbon atoms. Successive additions of methylene groups to the pendant chain made a fairly constant contribution to lowering the glass transition temperature. For pendant chains of four or more methylene groups, the rate of enthalpy relaxation was independent of the number of methylene groups in the pendant chain. The enthalpy relaxation data were fitted to the Cowie-Ferguson model and the relaxation times obtained were about 90 min. Dynamic mechanical analysis was employed to study the viscoelastic properties. The available observations indicate that the polymers become more flexible with increasing length of the pendant chain. The results suggest that the length of the pendant chain can be used effectively to control important material properties in this series of polymers.

Surface characterization of tyrosine-derived polycarbonates

The surfaces of five biodegradable tyrosine-derived polycarbonates were studied using contact angle measurements, ESCA, and static SIMS. The wettability, critical surface tension, and polarity of these polymers decreased with increasing chain length of the pendent alkyl groups. Surface elemental composition, as determined by ESCA, was consistent with the stoichiometry of the repeat unit of the polymers. High-resolution C1s, O1s, and N1s ESCA spectra also showed results consistent with the different bonding states of these elements in the polymer repeat unit. In both positive and negative ion spectra, SIMS experiments showed fragment ions characteristic of the polymer backbone. Fragment ions characteristic of the pendent groups were identified in the negative ion SIMS spectra only, while the positive SIMS spectra provided a characteristic fingerprint for each polymer.

AN EXPOSURE SYSTEM TO STUDY THE EFFECTS OF WATER-SOLUBLE GASES ON PM-INDUCED TOXICITY

An aerosol generation and exposure system to evaluate the role of water-soluble gases in particulate matter (PM)-induced injury was designed, built, and validated by generating test atmospheres to study the role of hydrogen peroxide in PM-induced toxicity. In this system, particle number concentration, size distribution, hydrogen peroxide concentration, and water concentration can all be varied. An ammonium sulfate aerosol with mass median diameter 0.46 +/- 0.01 microm was used as a model atmospheric aerosol because ammonium sulfate is a major component of the fine aerosol, and the water uptake of ammonium sulfate aerosol is well characterized. The following four test atmospheres were generated: (1) ammonium sulfate aerosol, (2) an aerosol containing hydrogen peroxide and ammonium sulfate, (3) vapor-phase hydrogen peroxide, and (4) particle-free air. All test atmospheres were maintained at a relative humidity of 85%. Particle size distribution, number concentration, total hydrogen peroxide concentration, temperature, and relative humidity were measured continuously in the exposure chamber. The gas-particle partitioning of hydrogen peroxide was calculated using total hydrogen peroxide concentration, the Henrys law constant for hydrogen peroxide in water, and aerosol water content. We found that the aerosol generation system produced stable concentrations throughout the 2-hour exposures.