Submucosal injection of poly(lactic-co-glycolic acid) microspheres in rabbit bladder as a potential treatment for urinary incontinence and vesicoureteral reflux: preliminary results

Endoscopic injection of bulking agents has been gaining attention as a therapy for urinary incontinence and vesicoureteral reflux because this therapy is simpler, less operation time-consuming and less painful than traditional surgical operations. The ideal bulking agent for the injection therapies must be easily injectable, biocompatible, volume-stable, non-antigenic and non-migratory. We evaluated poly(lactic-co-glycolic acid) (PLGA) microspheres as an injectable bulking agent for urologic injection therapies. To determine whether PLGA microspheres meet the requirements of an ideal bulking agent, PLGA microspheres were injected into the submucosal sites of a rabbit bladder wall. The microspheres were easily injectable. Two and five weeks post-implantation, histological examinations indicated that host cells from the surrounding bladder tissues migrated to the space between the injected microspheres and formed new hybrid tissue structures. Lymphocyte migration was noted around the implanted microspheres, but the inflammatory reaction diminished at 5 weeks. The hybrid tissue volume did not significantly decrease over time. There was no evidence of microsphere migration to the distant organs. Although long-term studies are needed to evaluate the therapeutic potential of this method, these preliminary results suggest the possibility of PLGA microspheres as a potentially useful injection material for urinary injection therapies.     

Poly(lactic-co-glycolic acid) microspheres as a potential bulking agent for urological injection therapy: preliminary results

Injection of bulking substances has been introduced as a new therapy to treat urinary incontinence and vesicoureteral reflux. Currently available bulking substances for the injection therapies include liquid or particulated silicone, collagen gel, and polytetrafluoroethylene paste. However, these materials have shown shortcomings such as inflammation, rapid volume decrease, and particle migration to distant organs. In the present study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microspheres as a potential injectable bulking agent for the injection therapies. PLGA microspheres (52 microm in average diameter) were injectable through various gauges of needles, as the injected microspheres showed no tendency to obstruct the needles and microsphere size exclusion was not observed upon injection through the needles. After injection of PLGA microspheres into the subcutaneous dorsum of mice, inflammation, new tissue volume change, and microsphere migration were examined. Host cells from the surrounding tissues migrated to the implanted microspheres and formed new hybrid tissue structures. The volume of the newly generated tissues was maintained approximately constant for 7 weeks. Histological analyses showed no evidence of migration of the implanted microspheres to the distant organs. In summary, PLGA microspheres were injectable and able to induce a new hybrid tissue formation without initial volume decrease or particle migration. These preliminary results suggest that this material may be a potentially useful bulking agent for urological injection therapies.     

The effect of microsphere degradation rate on the efficacy of polymeric microspheres as bulking agents: an 18-month follow-up study

The injection of bulking substances has been proposed as a new therapy to treat urinary incontinence and vesicoureteral reflux. Our previous study demonstrated that poly(lactic-co-glycolic acid) (PLGA) microspheres have the potential to serve as a bulking agent for urological injection therapies. Hybrid tissues exhibiting a bulking effect were formed in vivo by PLGA microsphere injection, but long-term volume stability was not proven. In this study, we hypothesized that the biodegradation rate of the bulking substance (polymer microspheres) would affect the duration of volume conservation of the induced hybrid tissue. To test this hypothesis, rapidly degrading 75:25 PLGA microspheres and slowly degrading poly(L-lactic acid) (PLLA) microspheres were used as injectable bulking agents for the injection therapy. In vitro degradation tests showed that the mass losses of PLLA and PLGA were 16 and 96% of the initial masses, respectively, at 30 weeks. PLLA and PLGA microspheres were injected into the subcutaneous dorsum of mice. Both types of microspheres were easily injectable through 24-gauge needles. Histological examinations at various time points indicated that host cells from the surrounding tissues migrated to the spaces between both types of injected microspheres and formed new hybrid tissue structures. Lymphocyte migration was noted around the implanted PLGA and PLLA microspheres, but the inflammatory reaction diminished with time. Importantly, the volume of the PLLA hybrid tissues slowly decreased to 52% of the initial volume at 12 months and maintained that volume until 18 months, whereas the volume of the PLGA hybrid tissues rapidly decreased to 22% at 6 months, and the PLGA hybrid tissues disappeared at 11 months. These results show that the biodegradation rate of the bulking substance may be useful for controlling the duration of volume conservation of the induced hybrid tissue.     

PCL microparticle-dispersed PLGA solution as a potential injectable urethral bulking agent

The PCL microparticle-dispersed PLGA solutions were prepared as a potential injectable urethral bulking agent. The mixture solutions were prepared by mixing polycarprolactone (PCL) microparticles (diameter, 100 approximately 200mum; fabricated by a temperature-induced phase transition method) and poly(dl-lactic-co-glycolic acid) (PLGA) solution (dissolved in tetraglycol to 10wt%) with different PCL microparticle to PLGA solution ratio. The mixture solution was solidified by the precipitation of PLGA when the solution was contact with water. In contact with water, the PCL microparticles exhibited a well-packed structure entrapped in a solidified porous PLGA matrix, which can effectively prevent the microparticle migration in the body and retain its initial volume even after PLGA matrix degradation. The PCL microparticle-dispersed PLGA solution (particle to solution ratio, 45/55 (w/v)) was easily injected through 18G needle into back of hairless mouse (subcutaneously) and stably located at the apply site. The surrounding tissue including blood vessel were gradually infiltrated into the implant up to 8 weeks without the initial injected volume change and with little inflammatory response. The PCL microparticle-dispersed PLGA solution may be a good candidate as an injectable bulking agent for the treatment of urinary incontinence owing to its good injectability, volume retention potential as well as biocompatibility.     

(Poly)acrylonitrile-based hydrogel as a therapeutic bulking agent in urology

Vesicoureteral reflux and urinary incontinence can be treated by endoscopic injection. Injectables such as collagen and hyaluronic acid-dextranomer are commonly used, however are not offering optimal therapeutic effect yet. In the present study a novel application for poly(acrylonitrile)-based hydrogel (Hypan), already tested in other clinical indications, has been investigated for its aptitude as an injectable bulking agent. This non-degradable, extremely hydrophilic hydrogel has the capacity of a 6-fold swelling once implanted into the body, assuring the bulking effect. Deposits of this bulking agent have been implanted into the submucosal space of the bladder of 6 minipigs and the histological reaction, as well as the physical implant behavior have been studied over a time period of 6 months. The implants have developed the desired bulking effect by the above-described hydrophilic swelling. They have been enclosed into a fine fibrous tissue capsule and provoked a very limited inflammatory reaction. This study has showed that poly(acrylonitrile)-based hydrogel implants injected into the bladder are well tolerated and, due to their defined bulking ability, feasible for the treatment of vesico-ureteral reflux and urinary incontinence. To determine the therapeutic efficiency of this treatment the implants will be studied in a vesicoureteral reflux and urinary incontinence animal model.     

Tissue engineered bulking agent with adipose-derived stem cells and silk fibroin microspheres for the treatment of intrinsic urethral sphincter deficiency

In this study we developed a tissue engineered bulking agent that consisted of adipose-derived stem cells (ADSCs) and silk fibroin microspheres to treat stress urinary incontinence caused by severe intrinsic sphincter deficiency (ISD). ISD models were established by completely transection of the bilateral pudendal nerve (PNT) and confirmed by the decreased leak-point pressure (LPP) and increased lumen area of urethra. Injection of silk fibroin microspheres could recover LPP and lumen area at 4 weeks but its efficacy disappears at 8, 12 weeks. Moreover, it was exciting to find that tissue engineered bulking agent brought long-term efficacy (at 4, 8, 12 weeks post-injection) on the recovery of LPP and lumen area. Concomitantly with the function, tissue engineered bulking agent treated group also improved the urethral sphincter structure as exhibited by better tissue regeneration. The findings showed that silk fibroin microspheres alone could work effectively in short-term, while tissue engineered bulking agent that combined silk fibroin microspheres with ADSCs exhibited promising long-term efficacy. This study developed a new strategy of tissue engineered bulking agent for future ISD therapy.     

Poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for cartilage tissue engineering

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.     

Tunable hydrogel-microsphere composites that modulate local inflammation and collagen bulking

Injectable biomaterials alone may alter local tissue responses, including inflammatory cascades and matrix production (e.g. stimulatory dermal fillers are used as volumizing agents that induce collagen production). To expand upon the available material compositions and timing of presentation, a tunable hyaluronic acid (HA) and poly(lactide-co-glycolide) (PLGA) microsphere composite system was formulated and assessed in subcutaneous and cardiac tissues. HA functionalized with hydroxyethyl methacrylate (HeMA) was used as a precursor to injectable and degradable hydrogels that carry PLGA microspheres (~50 μm diameter) to tissues, where the HA hydrogel degradation (~20 or 70 days) and quantity of PLGA microspheres (0-300 mgml(-1)) are readily varied. When implanted subcutaneously, faster hydrogel degradation and more microspheres (e.g. 75 mgml(-1)) generally induced more rapid tissue and cellular interactions and a greater macrophage response. In cardiac applications, tissue bulking may be useful to alter stress profiles and to stabilize the tissue after infarction, limiting left ventricular (LV) remodeling. When fast degrading HeMA-HA hydrogels containing 75 mgml(-1) microspheres were injected into infarcted tissue in sheep, LV dilation was limited and the thickness of the myocardial wall and the presence of vessels in the apical infarct region were increased ~35 and ~60%, respectively, compared to empty hydrogels. Both groups decreased volume changes and infarct areas at 8 weeks, compared to untreated controls. This work illustrates the importance of material design in expanding the application of tissue bulking composites to a range of biomedical applications.     

Development of new injectable bulking agents: biocompatibility of radiopaque polymeric microspheres studied in a mouse model

Radiopaque polymeric microspheres have a potential as new bulking agents for treatment of stress urinary incontinence (SUI). The advantage over existing bulking agents lies in their X-ray visibility in situ; other polymeric bulking agents (e.g., PTFE or silicone rubbers) are practically radiolucent (i.e., incapable of absorbing X-radiation). Radiopacity is useful in practice because of the high spatial accuracy of X-ray imaging. For instance, X-ray fluoroscopy can be used to assess possible migration of the bulking agent over time or to provide guidance in cases in which a second injection of a bulking agent is necessary (repeated treatment of SUI). Biocompatibility of injected radiopaque microspheres was investigated in vivo by using the mouse as a model. Microspheres were injected subcutaneously (9 animals) or intramuscularly (9 animals), and follow-up was 8 days or 3 months. X-ray fluoroscopy gave clear images of the microspheres as an ensemble, and it was found that no migration occurred during 3 months. Histopathology confirmed that all microspheres stayed close to the site of the injection. The microspheres appeared to be well tolerated; only a few giant cells, manifesting a mild inflammatory reaction, were encountered. At 3 months, capillary blood vessels were observed throughout the microsphere beds, and macrophages and fibroblast cells were seen in between the microspheres. This is encouraging with respect to the intended application, although it must be acknowledged that the data refer merely to a mouse model. Further experiments with larger, more representative models (rabbit and goat) are in progress.     

Bone response to fast-degrading, injectable calcium phosphate cements containing PLGA microparticles

Apatitic calcium phosphate cements (CPC) are frequently used to fill bone defects due to their favourable clinical handling and excellent bone response, but their lack of degradability inhibits complete bone regeneration. In order to render these injectable CaP cements biodegradable, hollow microspheres made of poly (D,L-lactic-co-glycolic) acid (PLGA) have been previously used as porogen since these microspheres were shown to be able to induce macroporosity upon degradation as well as to accelerate CPC degradation by release of acid degradation products. Recently, the capacity of PLGA microspheres to form porosity in situ in injectable CPCs was optimized by investigating the influence of PLGA characteristics such as microsphere morphology (dense vs. hollow) and end-group functionalization (acid terminated vs. end-capped) on acid production and corresponding porosity formation in vitro. The current study has investigated the in vivo bone response to CPCs containing two types of microspheres (hollow and dense) made of PLGA with two different end-group functionalizations (end capped and acid terminated). Microspheres were embedded in CPC and injected in the distal femoral condyle of New Zealand White Rabbits for 6 and 12 weeks. Histological results confirmed the excellent biocompatibility and osteoconductivity of all tested materials. Composites containing acid terminated PLGA microspheres displayed considerable porosity and concomitant bone ingrowth after 6 weeks, whereas end capped microspheres only revealed open porosity after 12 weeks of implantation. In addition, it was found that dense PLGA microspheres induced significantly more CPC degradation and bone tissue formation compared to hollow PLGA microspheres. In conclusion, it was shown that PLGA microspheres have a strong capacity to induce fast degradation of injectable CPC and concomitant replacement by bone tissue by controlled release of acid polymeric degradation products without compromising the excellent biocompatibility and osteoconductivity of the CPC matrix.     

The use of poly(lactic-co-glycolic acid) microspheres as injectable cell carriers for cartilage regeneration in rabbit knees

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.     

The use of poly(lactic-co-glycolic acid) microspheres as injectable cell carriers for cartilage regeneration in rabbit knees

The use of injectable scaffolding materials for in vivo tissue regeneration has raised great interest because it allows cell implantation through minimally invasive surgical procedures. Previously, we showed that poly(lactic-co-glycolic acid) (PLGA) microspheres can be used as an injectable scaffold to engineer cartilage in the subcutaneous space of athymic mice. The purpose of this study was to determine whether PLGA microspheres can be used as an injectable scaffold to regenerate hyaline cartilage in the osteochondral defects of rabbit knees. A full-thickness wound to the patellar groove of the articular cartilage was made in the knees of rabbits. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into these osteochondral wounds. Both chondrocyte transplantations without PLGA microspheres and culture medium injections without chondrocytes served as controls. Sixteen weeks after implantation, chondrocytes implanted using the PLGA microspheres formed white cartilaginous tissues. Histological scores indicating the extent of the cartilaginous tissue repair and the absence of degenerative changes were significantly higher in the experimental group than in the control groups (P < 0.05). Histological analysis by a hematoxylin and eosin stain of the group transplanted with microspheres showed thicker and better-formed cartilage compared to the control groups. Alcian blue staining and Masson's trichrome staining indicated a higher content of the major extracellular matrices of cartilage, sulfated glycosaminoglycans and collagen in the group transplanted with microspheres than in the control groups. In addition, immunohistochemical analysis showed a higher content of collagen type II, the major collagen type in cartilage, in the microsphere transplanted group compared to the control groups. In the group transplanted without microspheres, the wounds were repaired with fibro-cartilaginous tissues. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for cartilage regeneration in a rabbit model of osteochondral wound repair.     

A comparative biocompatibility study of microspheres based on crosslinked dextran or poly(lactic-co-glycolic)acid after subcutaneous injection in rats

Microspheres based on methacrylated dextran (dex-MA), dextran derivatized with lactate-hydroxyethyl methacrylate (dex-lactate-HEMA) or derivatized with HEMA (dex-HEMA) were prepared. The microspheres were injected subcutaneously in rats and the effect of the particle size and network characteristics [initial water content and degree of methacrylate substitution (DS)] on the tissue reaction was investigated for 6 weeks. As a control, poly(lactic-co-glycolic)acid (PLGA) microspheres with varying sizes (unsized, smaller than 10 microm, smaller and larger than 20 microm) were injected as well. A mild tissue reaction to the PLGA microspheres was observed, characterized by infiltration of macrophages (M?s) and some granulocytes. Six weeks postinjection, the PLGA microspheres were still present. However, their size was decreased indicating degradation and many spheres had been phagocytosed. The tissue reaction was hardly affected by size differences, except for particles smaller than 10 microm, which induced an extensive tissue reaction. The initial tissue reaction to nondegradable dex-MA microspheres was stronger than towards the PLGA microspheres, but at day 10 the tissue reactions were comparable for both groups. Six weeks postinjection, the dex-MA microspheres were completely phagocytosed, and no signs of degradation were observed. The size and initial water content of dex-MA microspheres hardly affected the tissue response, although less granulocytes were observed for microspheres with higher DS. Slowly degrading dextran microspheres composed of dex-(lactate(1)-)HEMA induced a tissue reaction comparable to the PLGA microspheres. However, degradation of the dex-(lactate(1,3)-)HEMA microspheres was associated with an increased number of M?'s and giant cells, both phagocytosing the microspheres and their degradation products. Similar to PLGA, no adverse reactions were observed for the nondegradable dex-MA and degradable dextran microspheres. This study shows that both nondegradable and degradable dextran-based microspheres are well tolerated after subcutaneous injection in rats, which make them interesting candidates as controlled drug delivery systems.     

Bioactive porous beads as an injectable urethral bulking agent: their in vitro evaluation on smooth muscle cell differentiation

Growth factor (basic fibroblast growth factor or vascular endothelial growth factor)-immobilized polycaprolactone (PCL)/Pluronic F127 porous beads were prepared as an injectable bulking agent for effective treatment of urinary incontinence. The growth factor-immobilized porous beads may stimulate smooth muscle cell (SMC) differentiation of muscle-derived stem cells or defect tissues around urethra to improve the sphincter function (bioactive therapy) as well as to provide a bulking effect (passive therapy). The porous PCL/F127 beads were fabricated by an isolated particle-melting/melt-molding particulate-leaching method. The growth factors were easily immobilized onto the surfaces of the PCL/F127 porous beads via heparin binding and were continuously released for up to 28 days. Both growth factor-immobilized porous beads had a positive effect for the SMC differentiation of muscle-derived stem cells, as were demonstrated by the analyses of quantitative polymerase chain reactions, Western blot using SMC-specific markers, and immunohistochemical staining. In particular, the basic fibroblast growth factor-immobilized porous beads showed desirable SMC differentiation behavior that can be applied as an injectable bulking agent for the treatment of urinary incontinence.     

Apatite-coated poly(lactic-co-glycolic acid) microspheres as an injectable scaffold for bone tissue engineering

Biodegradable polymer/ceramic composite scaffold could overcome limitations of biodegradable polymers or ceramics for bone regeneration. Injectable scaffold has raised great interest for bone regeneration in vivo, since it allows one for easy filling of irregularly shaped bone defects and implantation of osteogenic cells through minimally invasive surgical procedures The purpose of this study was to determine whether apatite-coated poly(lactic-co-glycolic acid) (PLGA) microspheres could be used as an injectable scaffold to regenerate bone in vivo. Apatite-coated PLGA microspheres were fabricated by incubating PLGA microspheres in simulated body fluid. The apatite that coated the PLGA microsphere surfaces was similar to apatite in natural bone, as demonstrated by scanning electron microscopy, X-ray diffraction spectra, energy-dispersive spectroscopy, and Fourier transformed-infrared spectroscopy analyses. Rat osteoblasts were mixed with apatite-coated PLGA microspheres and injected immediately into subcutaneous sites of athymic mice. Osteoblast transplantation with plain PLGA microspheres served as a control. Histological analysis of the implants at 6 weeks with hematoxylin and eosin staining, Masson's trichrome staining, and von Kossa staining revealed much better regeneration of bone in the apatite-coated PLGA microsphere group than the plain PLGA microsphere group. The new bone formation area and the calcium content of the implants were significantly higher in the apatite-coated PLGA microsphere group than in the plain PLGA microsphere group. This study demonstrates the feasibility of using apatite-coated PLGA microspheres as an injectable scaffold for in vivo bone tissue engineering. This scaffold may be useful for bone regeneration through minimally invasive surgical procedures in orthopedic applications.     

Controlled drug release from a novel injectable biodegradable microsphere/scaffold composite based on poly(propylene fumarate)

The ideal biomaterial for the repair of bone defects is expected to have good mechanical properties, be fabricated easily into a desired shape, support cell attachment, allow controlled release of bioactive factors to induce bone formation, and biodegrade into nontoxic products to permit natural bone formation and remodeling. The synthetic polymer poly(propylene fumarate) (PPF) holds great promise as such a biomaterial. In previous work we developed poly(DL-lactic-co-glycolic acid) (PLGA) and PPF microspheres for the controlled delivery of bioactive molecules. This study presents an approach to incorporate these microspheres into an injectable, porous PPF scaffold. Model drug Texas red dextran (TRD) was encapsulated into biodegradable PLGA and PPF microspheres at 2 microg/mg microsphere. Five porous composite formulations were fabricated via a gas foaming technique by combining the injectable PPF paste with the PLGA or PPF microspheres at 100 or 250 mg microsphere per composite formulation, or a control aqueous TRD solution (200 microg per composite). All scaffolds had an interconnected pore network with an average porosity of 64.8 +/- 3.6%. The presence of microspheres in the composite scaffolds was confirmed by scanning electron microscopy and confocal microscopy. The composite scaffolds exhibited a sustained release of the model drug for at least 28 days and had minimal burst release during the initial phase of release, as compared to drug release from microspheres alone. The compressive moduli of the scaffolds were between 2.4 and 26.2 MPa after fabrication, and between 14.9 and 62.8 MPa after 28 days in PBS. The scaffolds containing PPF microspheres exhibited a significantly higher initial compressive modulus than those containing PLGA microspheres. Increasing the amount of microspheres in the composites was found to significantly decrease the initial compressive modulus. The novel injectable PPF-based microsphere/scaffold composites developed in this study are promising to serve as vehicles for controlled drug delivery for bone tissue engineering.     

Adipose tissue engineering using mesenchymal stem cells attached to injectable PLGA spheres

The reconstruction of soft tissue defects remains a challenge in plastic and reconstructive surgery, and a real clinical need exists for an adequate solution. This study was undertaken in order to differentiate mesenchymal stem cells (MSCs) into adipocytes, and to then assess the possibility of constructing adipose tissue via the attachment of MSCs to injectable PLGA spheres. We also designed injectable PLGA spheres for scar-free transplantation. In this study, MSCs and adipo-MSCs (MSCs cultured in adipogenic medium for 7 days) were attached to PLGA spheres and cultured for 7 days, followed by injection into nude mice for 2 weeks. As a result, the difference between lipid accumulation in adipo-MSCs at 1 and 7 days was much higher in vitro than in the MSCs. Two weeks after injection, a massive amount of new tissue was formed in the APLGA group, whereas only a small amount was formed in the MPLGA group. We verified that the newly formed tissue originated from the injected MSCs via GFP testing, and confirmed that the created tissue was actual adipose tissue by oil red O staining and Western blot (PPAR(gamma) and C/EBP(alpha) were expressed only in APLGA groups). Therefore, this study presents an efficient model of adipose tissue engineering using MSCs and injectable PLGA spheres.     

Highly open porous biodegradable microcarriers: in vitro cultivation of chondrocytes for injectable delivery

Injectable cell therapy would provide a patient-friendly procedure for treatment of degenerated or wounded tissue. Biodegradable injectable porous microspheres were fabricated to use as dual-purpose microcarriers for cell culture and injectable scaffold for tissue regeneration. Gas foaming in a water-in-oil-in-water double emulsion was performed for fabricating the well-interconnected porous microcarriers using poly(lactic-co-glycolic acid) (PLGA). The gas foaming conditions were finely tuned to control the structural and morphological characteristics. Porous microcarriers with a mean size of approximately 175 microm and an average pore diameter of approximately 29 microm were produced for cell cultivation and injectable delivery. To promote cell seeding, amine-functionalized porous microcarriers were prepared by blending amine-functionalized PLGA with unreacted PLGA. To assess the porous microcarriers for chondrocyte cultivation, bovine articular chondrocytes were seeded and cultured in vitro in spinner flasks for 4 weeks. Visualization and biochemical analyses of the microcarrier-cell constructs were performed to demonstrate cell proliferation and phenotypic expression. Quantification of deoxyribonucleic acid, glycosaminoglycan, and collagen content showed that much greater cell proliferation and expression of cartilage-specific phenotype were observed for cultures in the following order: amine-functionalized porous microcarriers, porous microcarriers, nonporous microcarriers, and monolayer culture.     

Biocompatibility of lipid-protein-sugar particles containing bupivacaine in the epineurium.

Novel lipid-protein-sugar particles (LPSPs) are potentially biocompatible because they are composed of naturally occurring ingredients and their expected tissue dwell times are relatively short. In this research, we used histological sections to study tissue reaction to LPSPs (4.4-microm median diameter) when used for sciatic nerve block in the rat. As a reference, we compared LPSPs to 60-microm median diameter poly(lactic-co-glycolic) acid (PLGA) microspheres (110,000 MW PLGA, glycolic/lactic ratio 65:35). Four days after injection, both particle types produced acute inflammation within the confines of the injectate, inflammation in adjacent tissues, and myotoxicity. Bupivacaine-free particles did not display myotoxicity, and inflammation in adjacent tissues was reduced. At 2 weeks, inflammation from LPSPs had almost disappeared, whereas PLGA microspheres had a foreign-body giant cell reaction until at least 8 weeks after injection. In contrast, 3.6-microm median diameter, 20,000-MW PLGA microspheres produced a primarily histiocytic reaction 2 weeks after injection. In summary, the LPSPs and PLGA microspheres studied herein have excellent biocompatibility, but tissue reaction to the former is of much shorter duration. Myotoxicity and inflammation of surrounding tissue is largely attributed to bupivacaine. Foreign-body giant cells may be attributed to particle size rather than a specific reaction to PLGA.     

Injectable calcium phosphate cement with PLGA, gelatin and PTMC microspheres in a rabbit femoral defect

In this study, we investigated the in vivo degradation properties and tissue response towards injectable calcium phosphate cement (CPC) with no further addition, or calcium phosphate composite cement containing approximately 50 vol.% of microspheres. Three types of spheres were assessed, i.e. poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL) and poly(trimethylene carbonate) (PTMC). The cements were injected into 4.6 mm diameter and 6mm deep cylindrical defects in the femoral condyle of New Zealand white rabbits, hardened in situ and, after wound closure, left to heal for 4, 8 and 12 weeks (n=6 for each composition and time period). After retrieval, specimens were analyzed using histological and histomorphometrical methods. Results showed that non-modified CPCs showed excellent bone contact but only very limited erosion at the surface. The CPC/PLGA implant degraded almost completely, while tissue response significantly improved at each time period. CPC/PTMC showed slower degradation characteristics compared to CPC/PLGA. Finally, at all time periods, there was an evident inflammatory response to the CPC/GEL composite cement. In conclusion, the degradation properties of the CPC/PLGA microspheres composite and its bone response when implanted into the femoral condyles of rabbits were significantly better than those of CPC/gelatin and CPC/PTMC microspheres composites.