Meniscal repair with additive manufacture of bioresorbable polymer: From physicochemical characterization to implantation of 3D printed poly (L-co-D, L lactide-co-trimethylene carbonate) with autologous stem cells in rabbits.

Three-dimensional (3D) structures are actually the state-of-the-art technique to create porous scaffolds for tissue engineering. Since regeneration in cartilage tissue is limited due to intrinsic cellular properties this study aims to develop and characterize three-dimensional porous scaffolds of poly (L-co-D, L lactide-co-trimethylene carbonate), PLDLA-TMC, obtained by 3D fiber deposition technique. The PLDLA-TMC terpolymer scaffolds (70:30), were obtained and characterized by scanning electron microscopy, gel permeation chromatography, differential scanning calorimetry, thermal gravimetric analysis, compression mechanical testing and study on in vitro degradation, which showed its amorphous characteristics, cylindrical geometry, and interconnected pores. The in vitro degradation study showed significant loss of mechanical properties compatible with a decrease in molar mass, accompanied by changes in morphology. The histocompatibility association of mesenchymal stem cells from rabbit's bone marrow, and PLDLA-TMC scaffolds, were evaluated in the meniscus regeneration, proving the potential of cell culture at in vivo tissue regeneration. Nine New Zealand rabbits underwent total medial meniscectomy, yielding three treatments: implantation of the seeded PLDLA-TMC scaffold, implantation of the unseeded PLDLA-TMC and negative control (defect without any implant). After 24 weeks, the results revealed the presence of fibrocartilage in the animals treated with polymer. However, the regeneration obtained with the seeded PLDLA-TMC scaffolds with mesenchymal stem cells had become intimal to mature fibrocartilaginous tissue of normal meniscus both macroscopically and histologically. This study demonstrated the effectiveness of the PLDLA-TMC scaffold in meniscus regeneration and the potential of mesenchymal stem cells in tissue engineering, without the use of growth factors. It is concluded that bioresorbable polymers represent a promising alternative for tissue regeneration.

Crosslinked Collagen-Hyaluronic Acid Scaffold Enhances Interleukin-10 Under Co-Culture of Macrophages And Adipose-Derived Stem Cells.

The skin, the human body's largest organ, possesses a protective barrier that renders it susceptible to various injuries, including burns. Following burn trauma, the inflammatory process triggers both innate and adaptive immune responses, leading to the polarization of macrophages into two distinct phenotypes: the pro-inflammatory M1 and the anti-inflammatory M2. This dual response sets the stage for wound healing and subsequent tissue regeneration. Contributing to this transition from M1 to M2 polarization are human adipose-derived stem cells (ASCs), which employ paracrine signaling and inflammation suppression to enhance the remodeling phase. ASCs, when combined with biocompatible polymers, can be integrated into functional scaffolds. This study introduces an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-crosslinked (EDC-crosslinked) collagen-hyaluronic acid (Col-HA) scaffold assembled with ASCs, designed as a natural biomaterial device to modulate macrophage behavior in vitro under co-culture conditions. This innovation aims to improve wound healing processes. The EDC-crosslinked Col-HA scaffold favored the release of anti-inflammatory cytokines by ASCs, which indicated the M2 prevalence. In tissue engineering, a critical objective lies in the development of functional biomaterials capable of guiding specific tissue responses, notably the control of inflammatory processes. Thus, this research not only presents original findings but also points toward a promising avenue within regenerative medicine.

Electrospun Membrane Based on Poly(L-co-D,L lactic acid) and Natural Rubber Containing Copaiba Oil Designed as a Dressing with Antimicrobial Properties.

Drug delivery systems of natural antimicrobial compounds, such as copaiba oil (CO), have become relevant in the scientific community due to the recent prevalence of the public health complications related to antibiotic resistance. Electrospun devices act as an efficient drug delivery system for these bioactive compounds, reducing systemic side effects and increasing the effectiveness of the treatment. In this way, the present study aimed to evaluate the synergistic and antimicrobial effect of the direct incorporation of different concentrations of CO in a poly(L-co-D,L lactic acid) and natural rubber (NR) electrospun membrane. It was observed that CO showed bacteriostatic and antibacterial effects against S. aureus in antibiogram assays. The prevention of biofilm formation was confirmed via scanning electron microscopy. The test with crystal violet demonstrated strong bacteria inhibition in membranes with 75% CO. A decrease in hydrophilicity, observed in the swelling test, presented that the addition of CO promotes a safe environment for the recovery of injured tissue while acting as an antimicrobial agent. In this way, the study showed strong bacteriostatic effects of the CO incorporation in combination with electrospun membranes, a suitable feature desired in wound dressings in order to promote a physical barrier with prophylactic antimicrobial properties to avoid infections during tissue healing.

In Vitro Cell Behavior and Antibiotic Activity under Sustained Release of Doxycycline-Loaded Poly(lactic-co-glycolic acid) Microspheres.

The state-of-the-art sustained drug delivery systems are related to features to improve pharmacological transport through a controlled ratio between drug release and the desired therapeutic effect. Microspheres of biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), play an important role in these approaches, directing the release in a specific region of interest. In this way, the encapsulation of doxycycline (DOX) as a microbial agent turns the PLGA microspheres into a potential device for the treatment of topic oral diseases. Thus, this work aimed to produce DOX-loaded PLGA microspheres and see how they interfered with mesenchymal stem cell viability and in the sustained release in antimicrobial assays. Scanning electron microscopy showed the spherical microstructured pattern, revealing assorted sized distribution, with major diameters ranging 1-3 µm. The encapsulation efficiency presented a mean of 80% in both methods to obtain the microspheres (sonication and magnetic rotation). The DOX release test revealed a gradual and continuous profile of 30-40% between 120 and 168 h. Mesenchymal stem cells cultured in PLGA with or without DOX at several concentrations revealed no effect on the cell metabolic activity. Striking morphology changes were observed by confocal microscopy after 1 to 3 days under culture. The live/dead assay indicated that when microsphere densities were increased (from 10 to 100 µg/mL) cultured cells presented an internalized pattern of microspheres in both groups of PLGA containing DOX or not, while slight cell death signals were identified nearby microsphere clusters. Microbiological assays performed by the agar diffusion test pointed out that an inhibition zone was identified in Staphylococcus aureus (S. aureus) cultures at earlier times of DOX release. Despite the well-known use of PLGA as a drug delivery vehicle, when synthesized with DOX, it presents both characteristics of the desired treatment to prevent healthy tissue damage while avoiding bacterial growth in a microenvironment with anatomical features, such as grooves, projections, and other tough conditions that favor the development of oral diseases.

Poly(L-co-D,L lactic acid-co-Trimethylene Carbonate) 3D printed scaffold cultivated with mesenchymal stem cells directed to bone reconstruction: In vitro and in vivo studies.

A recent and quite promising technique for bone tissue engineering is the 3D printing, peculiarly regarding the production of high-quality scaffolds. The 3D printed scaffold strictly provides suitable characteristics for living cells, in order to induce treatment, reconstruction and substitution of injured tissue. The purpose of this work was to evaluate the behavior of the 3D scaffold based on Poly(L-co-D,L lactic acid-co-Trimethylene Carbonate) (PLDLA-TMC), which was designed in Solidworks™ software, projected in 3D Slicer™, 3D printed in filament extrusion, cultured with mesenchymal stem cells (MSCs) and tested in vitro and in vivo models. For in vitro study, the MSCs were seeded in a PLDLA-TMC 3D scaffold with 600 µm pore size and submitted to proliferation and osteogenic differentiation. The in vivo assays implanted the PLDLA-TMC scaffolds with or without MSCs in the calvaria of Wistar rats submitted to 8 mm cranial bone defect, in periods of 8-12 weeks. The results showed that PLDLA-TMC 3D scaffolds favored adherence and cell growth, and suggests an osteoinductive activity, which means that the material itself augmented cellular differentiation. The implanted PLDLA-TMC containing MSCs, showed better results after 12 weeks prior grafting, due the absence of inflammatory processes, enlarged regeneration of bone tissue and facilitated angiogenesis. Notwithstanding, the 3D PLDLA-TMC itself implanted enriched tissue repair; the addition of cells known to upregulate tissue healing reinforce the perspectives for the PLDLA-TMC applications in the field of bone tissue engineering in clinical trials.

Optimized Method to Improve Cell Activity in 3D Scaffolds Under a Dual Real-Time Dynamic Bioreactor System.

Bioreactor systems that allow the simulation of in vivo variables in a controlled in vitro environment, were a great advance in the field of tissue engineering. Due to the dynamic-mechanical features that some tissues present, 3D-engineered constructs often do not exhibit the biomechanical properties of these native tissues. Thus, a successful approach must not only achieve tissue repair but also restore its function after injury. Here, we describe a method to improve cell activity in 3D scaffolds in a dynamic bioreactor system through the application of mechanical compression and fluid flow for tissue engineering approaches.

A New Dermal Substitute Containing Polyvinyl Alcohol with Silver Nanoparticles and Collagen with Hyaluronic Acid: In Vitro and In Vivo Approaches.

The experimental use of poly (alcohol-vinyl) (PVA) as a skin curative is increasing widely. However, the use of this hydrogel is challenging due to its favorable properties for microbiota growth. The association with silver nanoparticles (AgNPs) as an antimicrobial agent turns the match for PVA as a dressing, as it focuses on creating a physical barrier to avoid wound dehydration. When associated with extracellular components, such as the collagen matrix, the device obtained can create the desired biological conditions to act as a skin substitute. This study aimed to analyze the anti-microbiological activity and the in vitro and in vivo responses of a bilaminar device of PVA containing AgNPs associated with a membrane of collagen-hyaluronic acid (col-HA). Additionally, mesenchymal stem cells were cultured in the device to evaluate in vitro responses and in vivo immunomodulatory and healing behavior. The device morphology revealed a porous pattern that favored water retention and in vitro cell adhesion. Controlled wounds in the dorsal back of rat skins revealed a striking skin remodeling with new epidermis fulfilling all previously injured areas after 14 and 28 days. No infections or significant inflammations were observed, despite increased angiogenesis, and no fibrosis-markers were identified as compared to controls. Although few antibacterial activities were obtained, the addition of AgNPs prevented fungal growth. All results demonstrated that the combination of the components used here as a dermal device, chosen according to previous miscellany studies of low/mid-cost biomaterials, can promote skin protection avoiding infections and dehydration, minimize the typical wound inflammatory responses, and favor the cellular healing responses, features that give rise to further clinical trials of the device here developed.

Enhancement of cartilage extracellular matrix synthesis in Poly(PCL-TMC)urethane scaffolds: a study of oriented dynamic flow in bioreactor.

The development of new technologies to produce three-dimensional and biocompatible scaffolds associated with high-end cell culture techniques have shown to be promising for the regeneration of tissues and organs. Some biomedical devices, as meniscus prosthesis, require high flexibility and tenacity and such features are found in polyurethanes which represent a promising alternative. The Poly(PCL-TMC)urethane here presented, combines the mechanical properties of PCL with the elasticity attributed by TMC and presents great potential as a cellular carrier in cartilage repair. Scanning electron microscopy showed the presence of interconnected pores in the three-dimensional structure of the material. The scaffolds were submitted to proliferation and cell differentiation assays by culturing mesenchymal stem cells in bioreactor. The tests were performed in dynamic flow mode at the rate of 0.4 mL/min. Laser scanning confocal microscopy analysis showed that the flow rate promoted cell growth and cartilage ECM synthesis of aggrecan and type II collagen within the Poly(PCL-TMC)urethane scaffolds. This study demonstrated the applicability of the polymer as a cellular carrier in tissue engineering, as well as the ECM was incremented only when under oriented flow rate stimuli. Therefore, our results may also provide data on how oriented flow rate in dynamic bioreactors culture can influence cell activity towards cartilage ECM synthesis even when specific molecular stimuli are not present. This work addresses new perspectives for future clinical applications in cartilage tissue engineering when the molecular factors resources could be scarce for assorted reasons.

In vitro and in vivo osteogenic potential of niobium-doped 45S5 bioactive glass: A comparative study.

In vitro and in vivo experiments were undertaken to evaluate the solubility, apatite-forming ability, cytocompatibility, osteostimulation, and osteoinduction for a series of Nb-containing bioactive glass (BGNb) derived from composition of 45S5 Bioglass. Inductively coupled plasma optical emission spectrometry (ICP-OES) revealed that the rate at which Na, Ca, Si, P, and Nb species are leached from the glass decrease with the increasing concentration of the niobium oxide. The formation of apatite as a function of time in simulated body fluid was monitored by 31P Magic Angle Spinning (MAS) Nuclear magnetic resonance spectroscopy. Results showed that the bioactive glasses: Bioglass 45S5 (BG45S5) and 1 mol%-Nb-containing-bioactive glass (BGSN1) were able to grow apatite layer on their surfaces within 3 h, while glasses with higher concentrations of Nb2 O5 (2.5 and 5 mol%) took at least 12 h. Nb-substituted glasses were shown to be compatible with bone marrow-derived mesenchymal stem cells (BMMSCs). Moreover, the bioactive glass with 1 mol% Nb2 O5 significantly enhanced cell proliferation after 4 days of treatment. Concentrations of 1 and 2.5 mol% Nb2 O5 stimulated osteogenic differentiation of BMMSCs after 21 days of treatment. For the in vivo experiments, trial glass rods were implanted into circular defects in rat tibia in order to evaluate their osteoconductivity and osteostimulation. Two morphometric parameters were analyzed: (a) thickness of new-formed bone layer and (b) area of new-formed subperiostal bone. Results showed that BGNb bioactive glass is osteoconductive and osteostimulative. Therefore, these results indicate that Nb-substituted glass is suitable for biomedical applications.

Alternative Cutaneous Substitutes Based on Poly(l-co-d,l-lactic acid-co-trimethylene carbonate) with Schinus terebinthifolius Raddi Extract Designed for Skin Healing.

The search for new therapies and drugs that act as topical agents to relieve pain and control the inflammatory processes in burns always attracted interest in clinical trials. As an alternative to synthetic drugs, natural extracts are useful in the development of new strategies and formulations for improving the quality of life. The aim of this study was to develop a wound dressing using poly(l-co-d,l-lactic acid-co-trimethylene carbonate) (PLDLA-TMC) containing Schinus terebinthifolius Raddi (S.T.R.). S.T.R. is a native Brazilian plant known for its strong anti-inflammatory responses. The membrane of PLDLA-TMC + S. terebinthifolius Raddi was prepared at different concentrations of S.T.R. (5, 10, 15, and 50%). The Fourier transform infrared results showed no change in the PLDLA-TMC spectrum after S.T.R. addition, whereas the swelling test showed changes only in PLDLA-TMC + S.T.R. at 50%. The wettability measurements showed a mass loss due to the decrease in the contact angle in all samples after the S.T.R. addition in the polymer, whereas the S.T.R. release test showed a linear delivery pattern. The scanning electron microscopy analysis showed that S.T.R. was homogeneously distributed at only 5 and 10%. Tensile tests demonstrated an increase in Young's modulus and a reduction in the elongation till rupture of PLDLA-TMC after the addition of S.T.R. The biocompatibility in vitro evaluation with rat fibroblast cells seeded in the membranes of PLDLA-TMC + S.T.R. showed that although S.T.R. interfered in cell morphology, all concentrations tested showed that cells were able to adhere and proliferate during 7 days. Thus, S.T.R. at 50% was chosen to be tested for in vivo trials. The histological and immunohistochemistry results revealed an accelerated skin healing at 7 days after controlled secondary burns were introduced in the dorsal skin, with a striking total recovery of the epidermis and high rates of molecular activation of cell proliferation. Due to the known biocompatibility properties of PLDLA-TMC and its stable release of S.T.R., we strongly recommend S.T.R.-containing PLDLA-TMC as a curative device to favor skin healing.

Impact of crystallinity and crystal size of nanostructured carbonated hydroxyapatite on pre-osteoblast in vitro biocompatibility.

Nanostructured carbonated hydroxyapatite (nCHA) is a promising biomaterial for bone tissue engineering due to its chemical properties, similar to those of the bone mineral phase and its enhanced in vivo bioresorption. However, the biological effects of nCHA nanoparticles on cells and tissues are not sufficiently known. This study assessed the impact of exposing pre-osteoblasts to suspensions with high doses of nCHA nanoparticles with high or low crystallinity. MC3T3-E1 pre-osteoblasts were cultured for 1 or 7 days in a culture medium previously exposed to CHA nanoparticles for 1 day. Control groups were produced by centrifugation for removal of bigger nCHA aggregates before exposure. Interaction of nanoparticles with the culture medium drastically changed medium composition, promoting Ca, P, and protein adsorption. Transmission Electron microscopy revealed that exposed cells were able to internalize both materials, which seemed concentrated inside endosomes. No cytotoxicity was observed for both materials, regardless of centrifugation, and the exposure did not induce alterations in the release of pro-and anti-inflammatory cytokines. Morphological analysis revealed strong interactions of nCHA aggregates with cell surfaces, however without marked alterations in morphological features and cytoskeleton ultrastructure. The overall in vitro biocompatibility of nCHA materials, regardless of physicochemical characteristics such as crystallinity, encourages further studies on their clinical applications.

Addition of Wollastonite Fibers to Calcium Phosphate Cement Increases Cell Viability and Stimulates Differentiation of Osteoblast-Like Cells.

Calcium phosphate cement (CPC) that is based on α-tricalcium phosphate (α-TCP) is considered desirable for bone tissue engineering because of its relatively rapid degradation properties. However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF) have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF). We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.

Thiamethoxam and picoxystrobin reduce the survival and overload the hepato-nephrocitic system of the Africanized honeybee.

Apis mellifera perform important pollination roles in agroecosystems. However, there is often intensive use of systemic pesticides in crops, which can be carried to the colony by forage bees through the collection of contaminated pollen and nectar. Inside the colony, pollen loads are stored by bees that add honey and several enzymes to this pollen. Nevertheless, intra-colonial chronic exposure could induce sublethal effects in young bees exposed to a wide range of pesticides present in these pollen loads. This study was aimed to both determine the survival rate and evaluate the sublethal effects on the hepato-nephrocitic system in response to continuous oral exposure to lower concentrations of neonicotinoid thiamethoxam (TXT) and picoxystrobin fungicide (PXT). Exposure to a single chemical and co-exposure to both pesticides were performed in newly emerged honeybee workers. A significant decrease in the bee survival rates was observed following exposure to TXT (0.001 ng a.i./µL) and PXT (0.018 ng a.i./µL), as well as following co-exposure to TXT+PXT/2. After five days of continuous exposure, TXT induced sub-lethal effects in the organs involved in the detoxification of xenobiotics, such as the fat body and pericardial cells, and it also induced a significant increase in the hemocyte number. Thus, the hepato-nephrocitic system (HNS) reached the greatest level of activity of pericardial cells as an attempt to eliminate this toxic compound from hemolymph. The HNS was activated at low levels by PXT without an increase in the hemocyte number; however, the mobilization of neutral glycoconjugates from the trophocytes of the fat body was prominent only in this group. TXT and PXT co-exposure induced intermediary morphological effects in trophocytes and pericardial cells, but oenocytes from the fat body presented with atypical cytoplasm granulation only in this group. These data showed that the realistic concentrations of these pesticides are harmful to newly emerged Africanized honeybees, indicating that intra-colonial chronic exposure drastically reduces the longevity of bees exposed to neonicotinoid insecticide (TXT) and the fungicide strobilurin (PXT) as in single and co-exposure. Additionally, the sublethal effects observed in the organs constituting the HNS suggest that the activation of this system, even during exposure to low concentrations of theses pesticides, is an attempt to maintain homeostasis of the bees. These data together are alarming because these pesticides can affect the performance of the entire colony.

Development of a membrane of poly (L-co-D,L lactic acid-co-trimethylene carbonate) with aloe vera: An alternative biomaterial designed to improve skin healing.

The search for new therapies and drugs that act as topical agents to relieve pain and control the infectious processes in burns always attracted interest in clinical trials. As an alternative to synthetic drugs, the use of natural extracts is useful in the development of new strategies and formulations for improving the life quality. The aim of this study was to develop a wound dressing using Poly(L-co-D,L lactic acid-co-TMC) (PLDLA-co-TMC) containing aloe vera (AV). This natural plant extract is known for its modulatory effects under healing process. The membrane of PLDLA-co-TMC+aloe vera was prepared at different concentrations of AV (5, 10, 15 and 50%). The FTIR showed no change in the PLDLA-co-TMC spectrum after AV addition, while the swelling test showed changes only in PLDLA-co-TMC+AV at 50%. The wettability measurements showed decrease in the contact angle in all samples after the AV addition in the polymer, while the AV release test showed that PLDLA-co-TMC+50%AV sample has higher AV release rate than the sample with other AV concentrations. The SEM analysis showed that AV was homogeneously distributed at 5% only. Tensile tests demonstrated an increase in the Young's modulus and a reduction in the elongation till rupture of the PLDLA-co-TMC after the addition of AV. Biocompatibility in vitro evaluation with fibroblast cells seeded in the membranes of PLDLA-co-TMC+AV showed that the cells were able to adhere, proliferate and maintain mitochondrial activity in all AV concentrations tested. Due to the known skin medicinal properties attributed to AV and the results here obtained, we suggest that after in vivo trials, the PLDLA-co-TMC+AV should be a promising biomaterial for application as a device for skin curative and healing agent.

Spotlight on Biomimetic Systems Based on Lyotropic Liquid Crystal.

The behavior of lyotropic biomimetic systems in drug delivery was reviewed. These behaviors are influenced by drug properties, the initial water content, type of lyotropic liquid crystals (LLC), swell ability, drug loading rate, the presence of ions with higher or less kosmotropic or chaotropic force, and the electrostatic interaction between the drug and the lipid bilayers. The in vivo interaction between LCC-drugs, and the impact on the bioavailability of drugs, was reviewed. The LLC with a different architecture can be formed by the self-assembly of lipids in aqueous medium, and can be tuned by the structures and physical properties of the emulsion. These LLC lamellar phase, cubic phase, and hexagonal phase, possess fascinating viscoelastic properties, which make them useful as a dispersion technology, and a highly ordered, thermodynamically stable internal nanostructure, thereby offering the potential as a sustained drug release matrix for drug delivery. In addition, the biodegradable and biocompatible nature of lipids demonstrates a minimum toxicity and thus, they are used for various routes of administration. This review is not intended to provide a comprehensive overview, but focuses on the advantages over non modified conventional materials and LLC biomimetic properties.

Study of mesenchymal stem cells cultured on a poly(lactic-co-glycolic acid) scaffold containing simvastatin for bone healing.

BACKGROUND: Tissue engineering is a promising alternative for the development of bone substitutes; for this purpose, three things are necessary: stem cells, a scaffold to allow tissue growth and factors that induce tissue regeneration. METHODS: To congregate such efforts, we used the bioresorbable and biocompatible polymer poly(lactic-co-glycolic acid) (PLGA) as scaffold. For the osteoinductive factor, we used simvastatin (SIM), a drug with a pleiotropic effect on bone growth. Mesenchymal stem cells (MSCs) were cultured in PLGA containing SIM, and the bone substitute of PLGA/SIM/MSC was grafted into critical defects of rat calvaria. RESULTS: The in vitro results showed that SIM directly interfered with the proliferation of MSC promoting cell death, while in the pure PLGA scaffold the MSC grew continuously. Scaffolds were implanted in the calvaria of rats and separated into groups: control (empty defect), PLGA pure, PLGA/SIM, PLGA/MSC and PLGA/SIM/MSC. The increase in bone growth was higher in the PLGA/SIM group. CONCLUSIONS: We observed no improvement in the growth of bone tissue after implantation of the PLGA/SIM/MSC scaffold. As compared with in vitro results, our main hypothesis is that the microarchitecture of PLGA associated with low SIM release would have created an in vivo microenvironment of concentrated SIM that might have induced MSC death. However, our findings indicate that once implanted, both PLGA/SIM and PLGA/MSC contributed to bone formation. We suggest that strategies to maintain the viability of MSCs after cultivation in PLGA/SIM will contribute to improvement of bone regeneration.

Bilaminar Device of Poly(Lactic-co-Glycolic Acid)/Collagen Cultured With Adipose-Derived Stem Cells for Dermal Regeneration.

Several materials are commercially available as substitutes for skin. However, new strategies are needed to improve the treatment of skin wounds. In this study, we developed and characterized a new device consisting of poly(lactic-co-glycolic acid) (PLGA) and collagen associated with mesenchymal stem cells derived from human adipose tissue. To develop the bilaminar device, we initially obtained a membrane of PLGA by dissolving the copolymer in chloroform and then produced a collagen type I scaffold by freeze-drying. The materials were characterized physically by gel permeation chromatography, scanning electron microscopy, and mass loss. Biological activity was assessed by cell proliferation assay. A preliminary study in vivo was performed with a pig model in which tissue regeneration was assessed macroscopically and histologically, the commercial device Integra being used as a control. The PLGA/collagen bilaminar material was porous, hydrolytically degradable, and compatible with skin growth. The polymer complex allowed cell adhesion and proliferation, making it a potentially useful cell carrier. In addition, the transparency of the material allowed monitoring of the lesion when the dressings were changed. Xenogeneic mesenchymal cells cultured on the device (PLGA/collagen/ASC) showed a reduced granulomatous reaction to bovine collagen, down-regulation of α-SMA, enhancement in the number of neoformed blood vessels, and collagen organization as compared with normal skin; the device was superior to other materials tested (PLGA/collagen and Integra) in its ability to stimulate the formation of new cutaneous tissue.

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 µgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

The impact of the RGD peptide on osteoblast adhesion and spreading on zinc-substituted hydroxyapatite surface.

The incorporation of zinc into the hydroxyapatite structure (ZnHA) has been proposed to stimulate osteoblast proliferation and differentiation. Another approach to improve cell adhesion and hydroxyapatite (HA) performance is coating HA with adhesive proteins or peptides such as RGD (arginine-glycine-aspartic acid). The present study investigated the adhesion of murine osteoblastic cells to non-sintered zinc-substituted HA disks before and after the adsorption of RGD. The incorporation of zinc into the HA structure simultaneously changed the topography of disk's surface on the nanoscale and the disk's surface chemistry. Fluorescence microscopy analyses using RGD conjugated to a fluorescein derivative demonstrated that ZnHA adsorbed higher amounts of RGD than non-substituted HA. Zinc incorporation into HA promoted cell adhesion and spreading, but no differences in the cell density, adhesion and spreading were detected when RGD was adsorbed onto ZnHA. The pre-treatment of disks with fetal bovine serum (FBS) greatly increased the cell density and cell surface area for all RGD-free groups, overcoming the positive contribution of zinc to cell adhesion. The presence of RGD on the ZnHA surface impaired the effects of FBS pre-treatment possibly due to competition between FBS proteins and RGD for surface binding sites.