Evaluation of a laminin-alginate biomaterial, adipocytes, and adipocyte-derived stem cells interaction in animal autologous fat grafting model using 7-Tesla magnetic resonance imaging.

Biomaterials are often added to autologous fat grafts both as supporting matrices for the grafted adipocytes and as cell carrier for adipose-derived stem cells (ADSCs). This in vivo study used an autologous fat graft model to test a lamininalginate biomaterial, adipocytes, and ADSCs in immune-competent rats. We transplanted different combinations of shredded autologous adipose tissue [designated "A" for adipose tissue]), laminin-alginate beads [designated "B" for bead], and ADSCs [designated "C" for cell]) into the backs of 15 Sprague-Dawley rats. Group A received only adipocytes, Group B received only laminin-alginate beads, Group AB received adipocytes mixed with laminin-alginate beads, Group BC received laminin-alginate beads encapsulating ADSCs, and Group ABC received adipocytes and laminin-alginate beads containing ADSCs. Seven-tesla magnetic resonance imaging was used to evaluate the rats at the 1st, 6th, and 12th weeks after transplantation. At the 12th week, the rats were sacrificed and the implanted materials were retrieved for gross examination and histological evaluation. The results based on MRI, gross evaluation, and histological data all showed that implants in Group ABC had better resorption of the biomaterial, improved survival of the grafted adipocytes, and adipogenic differentiation of ADSCs. Volume retention of grafts in Group ABC (89%) was also significantly greater than those in Group A (58%) (p < 0.01). Our findings support that the combination of shredded adipose tissue with ADSCs in laminin-alginate beads provided the best overall outcome.

In vitro and in vivo assessment of chitosan modified urocanic acid as gene carrier.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.

Laminin-Alginate Beads as Preadipocyte Carriers to Enhance Adipogenesis In Vitro and In Vivo.

The use of autologous fat grafting in breast reconstruction still requires optimization. Fat survival and calcification are the main issues that affect the outcomes of the procedure. In this study, a cell-based therapy utilizing laminin-alginate beads (LABs) as carriers was proposed to promote cell survival and adipogenesis by providing short-term physical support and facilitate nutrient diffusion of the implants. Laminin-modified alginate beads were fabricated by immobilizing laminin onto ring-opened alginate, used to encapsulate 3T3-L1 preadipocytes, and evaluated in vitro and in vivo. LABs as preadipocyte carriers showed better biocompatibility and stability than unmodified alginate beads. Preadipocytes in LABs had higher survival rate and enhanced adipogenesis than those in unmodified alginate beads. In vivo studies showed that LABs gradually degraded and the sites were replaced by newly formed fat tissues, and new blood vessels were also observed. 7T-MRI study mimicking clinical fat grafting showed that LABs carrying adipose stem cells improved the results of conventional fat grafts. Therefore, we believe that LABs represent promising cell carriers and can be potentially used for the reconstruction of breasts or other soft tissues in the future.

Modifying alginate with early embryonic extracellular matrix, laminin, and hyaluronic acid for adipose tissue engineering.

Extracellular matrix provides both mechanistic and chemical cues that can influence cellular behaviors such as adhesion, migration, proliferation, and differentiation. In this study, a new material, HA-L-Alg, was synthesized by linking developmentally essential ECM constituents hyaluronic acid (HA) and laminin(L) to alginate (Alg). The fabrication of HA-L-Alg was confirmed by FTIR spectroscopy, and it was used to form 3D cell-carrying beads. HA-L-Alg beads had a steady rate of degradation and retained 63.25% of mass after 9 weeks. HA-L-Alg beads showed biocompatibility comparable to beads formed by Alg-only with no obvious cytotoxic effect on the embedded 3T3-L1 preadipocytes. HA-L-Alg encapsulated 3T3-L1 cells were found to have a higher proliferation rate over those in Alg-only beads. These cells also showed better differentiation capacity after 2 weeks of adipogenic induction within HA-L-Alg beads. These results support that HA-L-Alg facilitated cell survival and proliferation, as well as stimulated and maintained cell differentiation. Our results suggest that HA-L-Alg has a great clinical potential to be used as stem cell carrier for adipose tissue engineering. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 669-677, 2016.

Insulin-loaded hydroxyapatite combined with macrophage activity to deliver insulin for diabetes mellitus.

We aimed to develop a diabetes mellitus (DM) treatment that could be administered by intramuscular (IM) injection and it lasted for more than 3 days. The objective was to load insulin into the lattice space of hydroxyapatite (HAP) to prevent its release based on a concentration gradient or detachment from the surface, with insulin release being aided by cellular activity. To avoid insulin denaturation during the synthesis of insulin-loaded HAP (insHAP), we developed a single-step insHAP synthesis method by the hydrolysis of brushite. X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) results suggested that insulin could be loaded into the HAP crystal lattice. After IM administration in rats with DM, the synthesized insHAP is thought to be engulfed by macrophages, escape from lysosome/endosome hybrids following disruption by osmotic pressure, and pumped into the extracellular space before entering the blood stream by diffusion. In rats with DM, the normal blood glucose level was maintained for 4 days after a single IM injection of the synthesized insHAP particles. Thus, insHAP may provide a breakthrough in insulin delivery for DM treatment, and may also be used to deliver fluorescent proteins, antibodies, and anticancer drugs.

Centrifugation versus PureGraft for fatgrafting to the breast after breast-conserving therapy.

BACKGROUND: Breast-conserving treatment (BCT) leads to a progressive and deteriorating breast deformity. Fatgrafting is ideal for breast reconstruction after BCT. The most frequently utilized technique for fat processing is centrifugation. The PureGraft device (Cytori Therapeutics, San Diego, CA, USA) is a new method that involves washing and filtering the fat to prepare the graft. We compared the subjective and objective outcomes of two fat-processing methods, centrifugation and PureGraft filtration. METHODS: Thirty patients underwent breast reconstruction performed by a single surgeon (OM) after BCT in our department between April 2011 and September 2012. The patients were preoperatively divided into two groups randomly: 15 received fatgrafts processed by centrifugation, and 15 received fatgrafts processed by washing in PureGraft bags. The patients were followed up for 12 to 30 months. To measure the subjective outcome, we distributed the BREAST-Q questionnaire to all the patients both preoperatively and 1 year postoperatively. The BCCT.core software evaluated the objective outcome of breast reconstruction by fatgrafting. RESULTS: The Breast-Q results indicated a tremendous improvement in the modules "Satisfaction with Breast" and "Psychosocial Well-being". The "Sexual Well-being" scale also improved. Only the module "Satisfaction with Breasts" significantly differed between groups; patients treated with the PureGraft fat exhibited better outcomes. The BCCT.core results did not significantly differ between the groups. CONCLUSION: One year postoperatively, the outcomes of the use of PureGraft bags or centrifugation to process fat for breast reconstruction after BCT did not differ. The unpredictability of the results following fatgrafting procedures is likely due to interindividual differences with yet-undisclosed causes.

Design and synthesis of elastin-like polypeptides for an ideal nerve conduit in peripheral nerve regeneration.

The study involves design and synthesis of three different elastin like polypeptide (ELP) gene monomers namely ELP1, ELP2 and ELP3 that encode for ELP proteins. The formed ELPs were assessed as an ideal nerve conduit for peripheral nerve regeneration. ELP1 was constructed with a small elongated pentapeptide carrying VPGVG sequence to mimic the natural polypeptide ELP. The ELP2 was designed by the incorporation of 4-penta peptide chains to improve the biocompatibility and mechanical strength. Thus, the third position in unique VPGVG was replaced with alanine to VPAVG and in a similar way modified to VPGKG, VPGEG and VPGIG with the substitution of lysine, glutamic acid and isoleucine. In ELP3, fibronectin C5 domain endowed with REDV sequence was introduced to improve the cell attachment. The ELP1, ELP2 and ELP3 proteins expressed by Escherichia coli were purified by inverse transition cycling (ITC). The purified ELPs were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The Schwann cell (SC) morphology and cell adhesion were assessed by fabrication of ELP membrane cross-linked with glutaraledhyde. The Schwann cell proliferation was measured by WST-1 assay. Immunofluorostaining of Schwann cells was accomplished with SC specific phenotypic marker, S100.

The in vivo performance of biomagnetic hydroxyapatite nanoparticles in cancer hyperthermia therapy.

Hyperthermia therapy for cancer has drawn more and more attention these days. In this study, we conducted an in vivo cancer hyperthermia study of the new magnetic hydroxyapatite nanoparticles by a mouse model. The magnetic hydroxyapatite nanoparticles were first made by co-precipitation method with the addition of Fe(2+). Then, magnetic-HAP powder (mHAP) or pure HAP powder (HAP) was mixed with phosphate buffer solution (PBS), respectively. The mixture was injected around the tumor. In order to achieve hyperthermia, the mice were placed into an inductive heater with high frequency and alternating magnetic field. Only the mice which were injected with mHAP and had been treated inside the magnetic field showed dramatic reduction of tumor volume, in the 15-day observation period. No local recurrence was noted. The blood test of mice proved that mHAP powders possessed good biocompatibility and little toxicity when injected subcutaneously. Therefore, our new magnetic hydroxyapatite nanoparticles have demonstrated therapeutic effect in a mouse model with little toxicity. Further study should be done before its application inside the human body.