Influence of electron-beam radiation on the hydrolytic degradation behaviour of poly(lactide-co-glycolide) (PLGA)

The purpose of this study is to examine the effect of electron-beam (e-beam) radiation on the hydrolytic degradation of poly(lactide-co-glycolide) (PLGA) films. PLGA films were irradiated and observed to undergo radiation-induced degradation through chain scission, as observed from a drop in its average molecular weight with radiation dose. Irradiated (5, 10 and 20 Mrad) and non-irradiated (0 Mrad) samples of PLGA were subsequently hydrolytically degraded in phosphate-buffered saline solution at 37.0 degrees C over a span of 12 weeks. It was observed that the natural logarithmic molecular weight (lnMn) of PLGA decreases linearly with hydrolytic degradation time. The rate of water uptake is higher for samples irradiated at higher radiation dose (e.g. 20 Mrad) and subsequently causing an earlier onset of mass loss. It is postulated that the increase in water uptake is due to the presence of more hydrophilic end groups, which results in the formation of microcavities because of an increase in osmotic pressure. A relationship between radiation dose and the rate of hydrolytic degradation of PLGA films, through its molecular weight was also established. This relationship allows a more accurate and precise control of the life span of PLGA through the use of e-beam radiation.     

聚乳酸、乳酸乙醇酸共聚物的制备及其体外降解

以外消旋乳酸、左旋乳酸和乙醇酸为原料,制备高纯度交酯单体丙交酯和乙交酯,以辛酸亚锡引发在高真空条件下本体熔融开环聚合,制得一系列不同分子量的聚乳酸均聚物和不同配比的乳酸乙醇酸共聚物,并用核磁共振氢谱、凝胶渗透色谱、差示扫描量热仪等手段对聚合产物的结构和性能进行分析表征.将溶液涂膜法制备的聚乳酸均聚物和乳酸乙醇酸共聚物薄膜置于Hank's人工模拟体液中降解试验,记录聚合物薄膜的失重和黏度及表面形貌变化,考察其体外降解规律,筛选适合作为冠脉支架表面载药涂层的聚合物,以推测其在人体内的降解行为.     

Guided bone regeneration by poly(lactic-co-glycolic acid) grafted hyaluronic acid bi-layer films for periodontal barrier applications

A novel protocol for the synthesis of biocompatible and degradation controlled poly(lactic-co-glycolic acid) grafted hyaluronic acid (HA-PLGA) was successfully developed for periodontal barrier applications. HA was chemically modified with adipic acid dihydrazide (ADH) in the mixed solvent of water and ethanol, which resulted in a high degree of HA modification up to 85 mol.%. The stability of HA-ADH to enzymatic degradation by hyaluronidase increased with ADH content in HA-ADH. When the ADH content in HA-ADH was higher than 80 mol.%, HA-ADH became soluble in dimethyl sulfoxide and could be grafted to the activated PLGA with N,N′-dicyclohexyl carbodiimide and N-hydroxysuccinimide. The resulting HA-PLGA was used for the preparation of biphasic periodontal barrier membranes in chloroform. According to in vitro hydrolytic degradation tests in phosphate buffered saline, HA-PLGA/PLGA blend film with a weight ratio of 1/2 degraded relatively slowly compared to PLGA film and HA coated PLGA film. Four different samples of a control, OSSIX^TM membrane, PLGA film, and HA-PLGA/PLGA film were assessed as periodontal barrier membranes for the calvarial critical size bone defects in SD rats. Histological and histomorphometric analyses revealed that HA-PLGA/PLGA film resulted in the most effective bone regeneration compared to other samples with a regenerated bone area of 63.1% covering the bone defect area.     

Polymers for biodegradable medical devices. VII. Hydroxybutyrate-hydroxyvalerate copolymers: degradation of copolymers and their blends with polysaccharides under in vitro physiological conditions

The hydrolytic degradation of hydroxybutyrate-hydroxyvalerate copolymers was monitored in vitro at 37 degrees C and pH 7.4. Direct use of bulk properties such as weight loss and tensile strength did not reveal substantial changes in the polymer matrix over degradation periods of several months. Despite this, the polymers were demonstrated to undergo significant modification during this period, in ways that markedly influence their subsequent behaviour. Combined use of goniophotometry and surface energy measurements revealed that surface modification begins at an early stage and is accompanied by diffusion of water into the matrix and a progressive increase in polymer porosity. Relatively little change in the molecular weight and some increase in the crystallinity of the matrix occurred during these early months. As a result, the tensile strength of the polymer varies little in this period. As the porosity of the matrix increases, hydrolytic chain scission within the matrix and diffusion out of degradation products proceeds more effectively. Decrease in matrix molecular weight, increase in matrix erosion, weight loss and loss of tensile strength began at a much more dramatic rate. The apparent resistance of the polymer to degradation in the early months is followed by an accelerated degradation phase around and beyond 1 yr. The use of filters that can dissolve or hydrolytically degrade more rapidly than the hydroxybutyrate matrix accelerates the development of porosity within the matrix and thus enhances the decomposition process.     

Effect of isothermal annealing on the hydrolytic degradation rate of poly(lactide-co-glycolide) (PLGA)

Isothermal crystallization through annealing at 115 degrees C was conducted to increase the degree of crystallinity of poly (lactide-co-glycolide) (PLGA). The maximum increase in the degree of crystallinity (approximately 21%) was achieved after 60 min of annealing. The crystal size/perfection was observed to increase with annealing time. The annealed PLGA films were then hydrolytically degraded in phosphate buffered saline solution of pH 7.4 at 37 degrees C for up to 150 days. Minimal mass loss was observed throughout the time investigated, suggesting that the samples were still in the first phase of degradation. The increase in the degree of crystallinity of the PLGA samples annealed at 15 and 30 min was found to retard their overall rate of hydrolytic degradation, when compared to those samples with higher initial crystallinity (annealed for 45 and 60 min) that had faster degradation rates. The increased degradation rate at higher crystallinity was associated with the loss of amorphous material and the formation of voids during annealing, which decreases the glass transition temperature and increases the average water uptake in the samples annealed for longer times. Therefore, the increase in degree of crystallinity is found to retard hydrolytic degradation but only to a certain extent, beyond which the formation of voids through annealing increases the rate of hydrolytic degradation.     

可吸收性生物材料PCL在体外与动物体内降解机制的研究

检测了可吸收性生物高分子材料ＰＣＬ在体外，动物体内的特性粘度，数均分子量Ｍｎ及结晶度的变化。探讨该材料在体外与动物体现降解的机制。试验结果表明，降解速率与材料原料分子量无关，降解的临界数均分子量（Ｍｎ）ｃｒ接近于５０００；在该分子量以前材料以水解机制为主并伴有表面酶浸蚀机制；在该分子量以后则以浸蚀机制为主，失重明显，并用扫描电镜图片比较了材料浸蚀前后的表面形态。     

Characterization of knitted polymeric scaffolds for potential use in ligament tissue engineering

Different scaffolds have been designed for ligament tissue engineering. Knitted scaffolds of poly-L-lactic acid (PLLA) yarns and co-polymeric yarns of PLLA and poly(glycolic acid) (PLGA) were characterized in the current study. The knitted scaffolds were immersed in medium for 20 weeks, before mass loss, molecular weight, pH value change in medium were tested; changes in mechanical properties were evaluated at different time points. Results showed that the knitted scaffolds had 44% porosity. There was no significant pH value change during degradation, while there was obvious mass loss at initial 4 week, as well as smooth molecular weight drop of PLLA. PLGA degraded more quickly, while PLLA kept its integrity for at least 20 weeks. Young's modulus increased while tensile strength and strain at break decreased with degradation time; however, all of them could maintain the basic requirements for ACL reconstruction. It showed that the knitted polymeric structures could serve as potential scaffolds for tissue-engineered ligaments.     

A novel polyethylene depot device for the study of PLGA and P(FASA) microspheres in vitro and in vivo

Polymer microspheres (0.5-5.0 microm) are difficult to characterize in vivo because they degrade, migrate, and are endocytosed. A novel polyethylene mesh pouch containing microspheres allowed for retrieval of degraded polymeric products from rats without affecting the rate of degradation. Pouches containing poly(lactic-co-glycolic acid) (PLGA) or poly(fumaric-co-sebacic acid) (P(FASA)) microspheres were implanted intramuscularly, subcutaneously, and intraperitoneally and analyzed after 3, 7, 14, and 28 days. In vivo, subcutaneous or intraperitoneal implants experienced an immediate mass loss and a delayed decrease in molecular weight (Mw). Intramuscular implants behaved similarly to in vitro samples, decreasing in Mw immediately and lagging in mass loss. These results suggest that mass loss, which is usually dependent on Mw loss in vitro, may be directly due to enzymatic, rather than hydrolytic, degradation subcutaneously and intraperitoneally, while intramuscular implants appear to be mostly dependent on hydrolytic cleavage. This observation is further supported by histology. Additional experiments on pouches loaded with PLGA microspheres encapsulating osteoprotegerin, a protein drug used to prevent bone resorption, revealed that use of the device prevented the artifactual polymer compression inherent to microsphere centrifugation during release studies and allowed for the extraction of active protein from microspheres implanted for 3 days in vivo.     

Nanomechanical properties of poly(lactic-co-glycolic) acid film during degradation

Despite the potential applications of poly(lactic-co-glycolic) acid (PLGA) coatings in medical devices, the mechanical properties of this material during degradation are poorly understood. In the present work, the nanomechanical properties and degradation of PLGA film were investigated. Hydrolysis of solvent-cast PLGA film was studied in buffer solution at 37 °C. The mass loss, water uptake, molecular weight, crystallinity and surface morphology of the film were tracked during degradation over 20 days. Characterization of the surface hardness and Young’s modulus was performed using the nanoindentation technique for different indentation loads. The initially amorphous films were found to remain amorphous during degradation. The molecular weight of the film decreased quickly during the initial days of degradation. Diffusion of water into the film resulted in a reduction in surface hardness during the first few days, followed by an increase that was due to the surface roughness. There was a significant delay between the decrease in the mechanical properties of the film and the decrease in the molecular weight. A sudden decline in mechanical properties indicated that significant bulk degradation had occurred.     

In vitro degradation of thin poly(DL-lactic-co-glycolic acid) films.

This study was designed to investigate the in vitro degradation of thin poly(DL-lactic-co-glycolic acid) (PLGA) films for applications in retinal pigment epithelium transplantation and guided tissue regeneration. PLGA films of copolymer ratios of 75:25 and 50:50 were manufactured with thickness levels of 10 microm (thin) and 100 microm (thick). Degradation of the films occurred during sample processing, and thin films with a higher surface area to volume ratio degraded faster. Sample weight loss, molecular weight loss, dimensional, and morphological changes were analyzed over a 10-week period of degradation in 0.2 M of phosphate-buffered saline (PBS), pH 7.4, at 37 degrees C. All PLGA films degraded by heterogeneous bulk degradation. Sample weights remained relatively constant for the first several weeks and then decreased dramatically. The molecular weights of PLGA films decreased immediately upon placement in PBS and continued to decrease throughout the time course. PLGA 50:50 films degraded faster than 75:25 films due to their higher content of hydrophilic glycolic units. The results also demonstrated that thick films degrade faster than corresponding thin films with the same composition. This was attributed to the greater extent of the autocatalytic effect, which further was confirmed by heterogeneous gel permeation chromatograms. These studies suggest that the degradation rate of thin films can be engineered by varying film thicknesses.     

Branched polyesters based on poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,L-lactide-co-glycolide): effects of polymer structure on in vitro degradation behaviour

Branched polyesters of the general structure poly[vinyl-3-(dialkylamino)alkylcarbamate-co-vinyl acetate-co-vinyl alcohol]-graft-poly(D,L-lactide-co-glycolide) have shown potential for nano- and micro-scale drug delivery systems. Here the in vitro degradation behaviour with a special emphasis on elucidating structure-property relationships is reported. Effects of type and degree of amine substitution as well as PLGA side chain length were considered. In a first set of experiment, the weight loss of solvent cast films of defined size from 19 polymers was measured as a function of incubation in phosphate buffer (pH 7.4) at 37 degrees C over a time of 21 days. A second study was initiated focusing on three selected polymers in a similar set up, but with additional observation of pH influences (pH 2 and pH 9) and determination of water uptake (swelling) and molecular weights during degradation. Scanning electron micrographs have been recorded at selected time points to characterize film specimens morphologically after degradation. Our investigations revealed the potential to influence the degradation of this polymer class by the degree of amine substitution, higher degrees leading to faster erosion. The erosion rate could further be influenced by the type of amine functionality, DEAPA-modified polyesters degrading as fast as or slightly faster than DMAPA-modified polyesters and these degrading faster than DEAEA-PVA-g-PLGA. As a third option the degradation rate could be modified by the PLGA side chain length, shorter side chains leading to faster erosion. As compared to linear PLGA, remarkably shorter degradation times could be achieved by grafting short PLGA side chains onto amine-modified PVA backbones. Erosion times from less than 5 days to more than 4 weeks could be realized by selecting the type of amine functionality, the degree of amine substitution and the PLGA side chain length at the time of synthesis. In addition, the pathway of hydrolytic degradation can be tuned to be either mainly bulk or surface erosion.     

Porous poly(alpha-hydroxyacid)/Bioglass composite scaffolds for bone tissue engineering. I: Preparation and in vitro characterisation

Highly porous composites scaffolds of poly-D,L-lactide (PDLLA) and poly(lactide-co-glycolide) (PLGA) containing different amounts (10, 25 and 50 wt%) of bioactive glass (45S5 Bioglass)were prepared by thermally induced solid-liquid phase separation (TIPS) and subsequent solvent sublimation. The addition of increasing amounts of Bioglass into the polymer foams decreased the pore volume. Conversely, the mechanical properties of the polymer materials were improved. The composites were incubated in phosphate buffer saline at 37 degrees C to study the in vitro degradation of the polymer by measurement of water absorption, weight loss as well as changes in the average molecular weight of the polymer and in the pH of the incubation medium as a function of the incubation time. The addition of Bioglass to polymer foams increased the water absorption and weight loss compared to neat polymer foams. However, the polymer molecular weight, determined by size exclusion chromatography, was found to decrease more rapidly and to a larger extent in absence of Bioglass. The presence of the bioactive filler was therefore found to delay the degradation rate of the polymer as compared to the neat polymer foams. Formation of hydroxyapatite on the surface of composites, as an indication of their bioactivity, was recorded by EDXA, X-ray diffractometry and confirmed by Raman spectroscopy.     

The modification of PLA and PLGA using electron-beam radiation

The degradable polymers polylactide (PLA) and polylactide-co-glycolide (PLGA) have found widespread use in modern medical practice. However, their slow degradation rates and tendency to lose strength before mass have caused problems. The aim of this study was to ascertain whether treatment with e-beam radiation could address these problems. Samples of PLA and PLGA were manufactured and placed in layered stacks, 8.1 mm deep, before exposure to 50 kGy of e-beam radiation from a 1.5 MeV accelerator. Gel permeation chromatography testing showed that the molecular weight of both materials was depth-dependent following irradiation, with samples nearest to the treated surface showing a reduced molecular weight. Samples deeper than 5.4 mm were unaffected. Computer modeling of the transmission of a 1.5 MeV e-beam in these materials corresponded well with these findings. An accelerated mass-loss study of the treated materials found that the samples nearest the irradiated surface initiated mass loss earlier, and at later stages showed an increased percentage mass loss. It was concluded that e-beam radiation could modify the degradation of bioabsorbable polymers to potentially improve their performance in medical devices, specifically for improved orthopedic fixation.     

Effect of blending calcium compounds on hydrolytic degradation of poly(DL-lactic acid-co-glycolic acid)

To clarify the effect of blending calcium compounds with different acidity or basicity on the degradation of poly(DL-lactic acid-co-glycolic acid) (PLGA). composite materials composed of PLGA incorporated with 30 mass% of calcium dihydrogenphosphate (CDHP), calcium hydrogenphosphate, calcium phosphate, and calcium carbonate (CC) were prepared by mixing in dioxane followed by freeze-drying. The porous composite materials were then compressed to yield nonporous films with 0.5 mm thickness. The blend films and the unfilled films as a control were immersed in phosphate-buffered saline (pH 7.4) at 37 degrees C. Specimens were removed at 1, 2, 3, 4, 6, and 8 weeks and subjected to measuring of water absorption, mass loss, thickness change, and molecular weight. The temporal changes in appearance and measurements for the five materials differed from each other; especially, the CC blend differed from the other four materials. The degradation of PLGA decreased with increasing basicity of the calcium compounds blended. The most basic CC was most effective to delay the degradation, while the most acidic CDHP was least effective. However, even CDHP had appreciable degradation-delaying effects compared with unfilled PLGA. Thus, the findings of the present study suggest that degradation of PLGA can be varied by blending inorganic compounds with different basicity.     

Hydrolytic and enzymatic incubation of polyhydroxyoctanoate (PHO): a short-term in vitro study of a degradable bacterial polyester

The present study examined the degradation behaviour of poly(beta-hydroxy octanoate) (PHO), a bacterial poly(beta-hydroxy alkanoate), following incubation under hydrolytic or enzymatic conditions in vitro. Solution-cast PHO films were incubated in a citrate buffer solution with and without acid phosphatase and in an acetate buffer with and without beta-glucuronidase for periods ranging from 7 to 60 days. The physical characterization of the PHO films was analyzed by SEM and tensile strength studies. In addition, various analytical methods were used to detect modifications in the chemical and morphological structure of the PHO, namely, ESCA, FTIR, DSC, X-ray diffraction, and SEC. The results indicate that the enzymatic conditions selected in the present study induced no significant surface morphological or chemical modifications, and no significant weight loss was observed after 60 days of incubation. However, as revealed by weight average molecular weight Mw and number average molecular weight Mn decreases, changes in the bulk structure of the PHO were observed with acid phosphatase at 28 and 60 days, in contrast to smaller Mw and Mn decreases recorded in both the buffers and the beta-glucuronidase. The tensile properties had decreased following incubation, yet showed no difference under all of the selected conditions. With no weight loss or surface changes, the PHO films incubated in acid phosphatase showed only a chemical hydrolytic process characterized by Mw and Mn decreases with time of incubation. The present study demonstrated that the degradation of PHO films is one of slow, chemical hydrolysis only, perhaps requiring several months of incubation. The hydrophobic nature of the long alkyl pendent chain in PHO may be responsible for this slow process. The inability of enzymes to degrade PHO may be attributed to the latter's poor adsorption capacity, due to its hydrophobic nature, and to a lack of specificity in the catalytic activity of these enzymes.     

Characterization of emulsified chitosan-PLGA matrices formed using controlled-rate freezing and lyophilization technique

This study evaluated the formation of chitosan-50:50 poly-lactic-co-glycolic acid (PLGA) blend matrices using controlled-rate freezing and lyophilization technique (CRFLT). An emulsion system was used in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), a cellular component, as a stabilizer. Blended scaffolds showed an open pore morphology and homogenous interdispersion of PLGA and chitosan. Forming emulsions after dissolving PLGA in chloroform, benzene, or methylene chloride indicated better emulsion stability with benzene and chloroform. Scaffolds formed by freezing at -20, -78, and -196 degrees C from these emulsions showed significant influence of the solvent and freezing temperature on the microarchitecture of the scaffold. By controlling the concentration of chitosan, scaffolds with greater than 90% porosity were attained. Since the two polymers degrade by different mechanisms, formed scaffolds were analyzed for degradation characteristics for 4 weeks in presence of 10 mg/L lysozyme. These results showed no significant difference in the weight loss and dimension changes, as all scaffolds showed significant (a) weight loss and (b) nearly 60% reduction in volume. Further, pH of the incubation media decreased in all the samples. When cellular activity of green fluorescence protein-transfected smooth muscle cells was analyzed, no apparent cytotoxicity was observed. However, the cell spreading area decreased. In summary, these results show promising potential in tissue engineering and drug delivery applications.     

Degradation of porous poly(D,L-lactic-co-glycolic acid) films based on water diffusion

Poly(D,L-lactic-co-glycolic acid) has been extensively used as a controlled release carrier for drug delivery due to its good biocompatibility, biodegradability, and mechanical strength. Effects of dense and porous film's degradation behavior have been systematically investigated up to 17 weeks in Hank's Simulated Body Fluid at 37 degrees C. The degradation of the films was studied by measuring changes in weight, molecular weight and its distribution, morphology, composition etc.. A special thing was that the differences in water diffusion in dense and porous structure films caused the different degradation behavior. According to the characteristic changes of various properties of films, the degradation process is suggested to be roughly divided into four stages, tentatively named as water absorption stage, dramatic loss of molecular weight or micro-pores formed stage, loss of weight or enlarged-pores formed stage, pores diminished or pores collapse stage.     

Intraocular degradation behavior of crosslinked and linear poly(trimethylene carbonate) and poly(D,L-lactic acid)

The intraocular degradation behavior of poly(trimethylene carbonate) (PTMC) networks and poly(D,L-lactic acid) (PDLLA) networks and of linear high molecular weight PTMC and PDLLA was evaluated. PTMC is known to degrade by enzymatic surface erosion in vivo, whereas PDLLA degrades by hydrolytic bulk degradation. Rod shaped specimens were implanted in the vitreous of New Zealand white rabbits for 6 or 13 wk. All materials were well tolerated in the rabbit vitreous. The degradation of linear high molecular weight PTMC and PTMC networks was very slow and no significant mass loss was observed within 13 wk. Only some minor signs of macrophage mediated erosion were found. The fact that no significant enzymatic surface erosion occurs can be related to the avascularity of the vitreous and the limited number of cells it contains. PDLLA samples showed more evident signs of degradation. For linear PDLLA significant swelling and a large decrease in molecular weight in time was observed and PDLLA network implants started to lose mass within 13 wk. Of the tested materials, PDLLA networks seem to be most promising for long term degradation controlled intravitreal drug delivery since this material degrades without significant swelling. Furthermore the preparation method of these networks allows easy and efficient incorporation of drugs.     

Microporous silk fibroin scaffolds embedding PLGA microparticles for controlled growth factor delivery in tissue engineering

The development of prototype scaffolds for either direct implantation or tissue engineering purposes and featuring spatiotemporal control of growth factor release is highly desirable. Silk fibroin (SF) scaffolds with interconnective pores, carrying embedded microparticles that were loaded with insulin-like growth factor I (IGF-I), were prepared by a porogen leaching protocol. Treatments with methanol or water vapor induced water insolubility of SF based on an increase in β-sheet content as analyzed by FTIR. Pore interconnectivity was demonstrated by SEM. Porosities were in the range of 70–90%, depending on the treatment applied, and were better preserved when methanol or water vapor treatments were prior to porogen leaching. IGF-I was encapsulated into two different types of poly(lactide-co-glycolide) microparticles (PLGA MP) using uncapped PLGA (50:50) with molecular weights of either 14 or 35 kDa to control IGF-I release kinetics from the SF scaffold. Embedded PLGA MP were located in the walls or intersections of the SF scaffold. Embedment of the PLGA MP into the scaffolds led to more sustained release rates as compared to the free PLGA MP, whereas the hydrolytic degradation of the two PLGA MP types was not affected. The PLGA types used had distinct effects on IGF-I release kinetics. Particularly the supernatants of the lower molecular weight PLGA formulations turned out to release bioactive IGF-I. Our studies justify future investigations of the developed constructs for tissue engineering applications.     

In vitro evaluation of biodegradation of poly(lactic-co-glycolic acid) sponges

Evaluation of the degradability of porous scaffolds is very important for tissue engineering. A protocol in which the condition is close to the in vivo pH environment was established for in vitro evaluation of biodegradable porous scaffolds. Degradation of PLGA sponges in phosphate-buffered solution (PBS) was evaluated with the protocol. The PLGA sponges degraded with incubation time. For the first 12 weeks, the weight loss increased gradually and then remarkably after 12 weeks. In contrast, the number-average molecular weight (Mn) decreased dramatically for the first 12 weeks and then less markedly after 12 weeks. Thermal analysis showed that the glass transition temperatures (Tg) decreased rapidly for the first 12 weeks, and the change became less evident after 12 weeks. These results suggest that the degradation mechanism of PLGA sponges was dominated by autocatalyzed bulk degradation for the first 12 weeks and then by surface degradation after 12 weeks. Physical aging was observed during incubation at 37 degrees C. The heterogeneous structure caused by physical aging might be one of the driving forces that induced autocatalyzed bulk degradation. The degradation mechanism was further supported by the data of pH change and the morphology of the degraded PLGA sponges. The autocatalyzed acidic products flooded out after 8 weeks, the pH dropped, and the walls of the sponges became more porous. The increase of the pore surface area facilitated surface degradation after 12 weeks. The pH was in the range between 7.43 and 7.24 during the entire incubation time. The protocol suppressed extreme changes of the pH and will be useful in the biodegradation evaluation of porous scaffolds for tissue engineering.