Incorporation of a Poly-ε-Caprolactone Scaffold in a ;Circular Stapled End-To-End Small Intestine Anastomosis Does Not Have Any Adverse Effects Within 30 days: A Study in Piglets.

Background. Incorporation of a poly-ε-caprolactone (PCL) scaffold in circular stapled anastomoses has been shown to increase the anastomotic tensile strength on postoperative day (POD) 5 in a pig model. The aim of this study was to investigate the effects of incorporation of a PCL scaffold in a circular stapled end-to-end small intestine anastomosis, with stricture formation and anastomotic histology as primary outcomes in a 30-day observation period. Methods. A total of 15 piglets were included. In each piglet, three circular stapled end-to-end anastomoses were made in the small intestines. Two were interventional and one was a control. On POD 10, 20, or 30, the anastomoses were subjected to in vivo intraluminal contrast study, and the index for anastomotic lumen was calculated. The anastomotic segment was resected and subjected to a tensile strength test and histological examination. Results. At POD 10, the mean ± SD value for anastomotic index was .749 ± .065 in control anastomoses and .637 ± .051 in interventional anastomosis (P = .0046), at POD 20, .541 ± .150 and .724 ± .07 (P = .051), and at POD 30, .645 ± .103 and .686 ± .057 (P = .341), respectively. No significant difference was observed in maximum tensile strength and histology at POD 30. Conclusions. The incorporation of a PCL scaffold in a circular stapled end-to-end small intestine anastomosis does not increase the risk of stricture or impair wound healing after 30 days.

Global MicroRNA Profiling in Human Bone Marrow Skeletal-Stromal or Mesenchymal-Stem Cells Identified Candidates for Bone Regeneration.

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of miRNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B (cyclin-dependent kinase inhibitor 1B). A number of additional miRNAs exerted additive osteoinductive effects on BMSC differentiation, suggesting that pools of miRNAs delivered locally from an implanted scaffold can provide a promising approach for enhanced bone regeneration.

Precipitant induced porosity augmentation of polystyrene preserves the chondrogenicity of human chondrocytes.

Cells constantly sense and receive chemical and physical signals from neighboring cells, interstitial fluid, and extracellular matrix, which they integrate and translate into intracellular responses. Thus, the nature of the surface on which cells are cultured in vitro plays an important role for cell adhesion, proliferation, and differentiation. Autologs chondrocyte implantation is considered the treatment of choice for larger cartilage defects in the knee. To obtain a sufficient number of chondrocytes for implantation multiple passaging is often needed, which raises concerns about the changes in the chondrogenic phenotype. In the present study, we analyzed the effect at cellular and molecular level of precipitant induced porosity augmentation (PIPA) of polystyrene surfaces on proliferation and differentiation of human chondrocytes. Human chondrocytes were isolated from healthy patients undergoing anterior cruciate ligament reconstruction and cultured on PIPA modified polystyrene surfaces. Microscopical analysis revealed topographically arranged porosity with micron pores and nanometer pits. Chondrocytes cultured on PIPA surfaces revealed no difference in cell viability and proliferation, but gene- and protein expressions of collagen type II were pronounced in the first passage of chondrocytes when compared to chondrocytes cultured on control surfaces. Additionally, an analysis of 40 kinases revealed that chondrocytes expanded on PIPA caused upregulated PI3K/mTOR pathway activation and inhibition of mTORC1 resulted in reduced sGAG synthesis. These findings indicate that PIPA modified polystyrene preserved the chondrogenicity of expanded human chondrocytes at gene and protein levels, which clinically may be attractive for the next generation of cell-culture surfaces for ex vivo cell growth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3073-3081, 2016.

Improvement of Distribution and Osteogenic Differentiation of Human Mesenchymal Stem Cells by Hyaluronic Acid and ß-Tricalcium Phosphate-Coated Polymeric Scaffold In Vitro.

Bone tissue engineering requires a well-designed scaffold that can be biodegradable, biocompatible, and support the stem cells to osteogenic differentiation. Porous polycaprolactone (PCL) scaffold prepared by fused deposition modeling is an attractive biomaterial that has been used in clinic. However, PCL scaffolds lack biological function and osteoinductivity. In this study, we functionalized the PCL scaffolds by embedding them with a matrix of hyaluronic acid/ß-tricalcium phosphate (HA/TCP). Human mesenchymal stem cells (MSCs) were cultured on scaffolds with and without coating to investigate proliferation and osteogenic differentiation. The DNA amount was significantly higher in the HA/TCP-coated scaffold on day 21. At the gene expression level, HA/TCP coating significantly increased the expression of ALP and COLI on day 4. These data correlated with the ALP activity peaking on day 7 in the HA/TCP-coated scaffold. Scanning electron microscope and histological analysis revealed that the cell matrix and calcium deposition were distributed more uniformly in the coated scaffolds compared to scaffolds without coating. In conclusion, the HA/TCP coating improved cellular proliferation, osteogenic differentiation, and uniform distribution of the cellular matrix in vitro. The HA/TCP-PCL scaffold holds great promise to accommodate human bone marrow-derived MSCs for bone reconstruction purposes, which warrants future in vivo studies.

Free radicals generated by tantalum implants antagonize the cytotoxic effect of doxorubicin.

Little is known about the interaction between antineoplastic drugs and implants in bone cancer patients. We investigated the interaction between commercially available porous tantalum (Ta) implants and the chemotherapeutic drug, Doxorubicin (DOX). DOX solutions were prepared in the presence of Ta implant. The changes in fluorescence intensity of the DOX chromophore were measured by spectrofluorometry and the efficacy of DOX was evaluated by viability of rabbit rectal tumor cells (VX2). After 5 min interaction of the DOX solution (5 µg/ml) with the Ta implant, the fluorescent intensity of the DOX solution was 85% degraded, and only 20% the drug efficacy to kill VX2 cells was retained. However, after adding a reducing agent, Dithiothreitol (DTT, 10 µg/ml), 80% of the original fluorescence and 50% of the drug efficacy were restored while UV irradiation enhanced drug degradation in the presence of Ta implant. The action of DTT and UV irradiation indicated that reactive oxygen species (ROS) were involved in the drug degradation mechanism. We detected that Ta implants in aqueous medium produced hydroxyl radicals. Cells showed higher intracellular ROS activity when culture medium was incubated with the Ta implant prior to cell culture. It is concluded that the porous Ta implant antagonizes the cytotoxicity of DOX via ROS generation of the porous Ta implant.

A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds.

We describe a simple method for bone engineering using biodegradable scaffolds with mesenchymal stem cells derived from human induced-pluripotent stem cells (hiPS-MSCs). The hiPS-MSCs expressed mesenchymal markers (CD90, CD73, and CD105), possessed multipotency characterized by tri-lineages differentiation: osteogenic, adipogenic, and chondrogenic, and lost pluripotency - as seen with the loss of markers OCT3/4 and TRA-1-81 - and tumorigenicity. However, these iPS-MSCs are still positive for marker NANOG. We further explored the osteogenic potential of the hiPS-MSCs in synthetic polymer polycaprolactone (PCL) scaffolds or PCL scaffolds functionalized with natural polymer hyaluronan and ceramic TCP (PHT) both in vitro and in vivo. Our results showed that these iPS-MSCs are functionally compatible with the two 3D scaffolds tested and formed typically calcified structure in the scaffolds. Overall, our results suggest the iPS-MSCs derived by this simple method retain fully osteogenic function and provide a new solution towards personalized orthopedic therapy in the future.

Fabrication and characterization of a rapid prototyped tissue engineering scaffold with embedded multicomponent matrix for controlled drug release.

Bone tissue engineering implants with sustained local drug delivery provide an opportunity for better postoperative care for bone tumor patients because these implants offer sustained drug release at the tumor site and reduce systemic side effects. A rapid prototyped macroporous polycaprolactone scaffold was embedded with a porous matrix composed of chitosan, nanoclay, and ß-tricalcium phosphate by freeze-drying. This composite scaffold was evaluated on its ability to deliver an anthracycline antibiotic and to promote formation of mineralized matrix in vitro. Scanning electronic microscopy, confocal imaging, and DNA quantification confirmed that immortalized human bone marrow-derived mesenchymal stem cells (hMSC-TERT) cultured in the scaffold showed high cell viability and growth, and good cell infiltration to the pores of the scaffold. Alkaline phosphatase activity and osteocalcin staining showed that the scaffold was osteoinductive. The drug-release kinetics was investigated by loading doxorubicin into the scaffold. The scaffolds comprising nanoclay released up to 45% of the drug for up to 2 months, while the scaffold without nanoclay released 95% of the drug within 4 days. Therefore, this scaffold can fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro. These results suggest that the scaffold can be used clinically in reconstructive surgery after bone tumor resection. Moreover, by changing the composition and amount of individual components, the scaffold can find application in other tissue engineering areas that need local sustained release of drug.

Navigated percutaneous lumbosacral interbody fusion: a feasibility study with three-dimensional surgical simulation and cadaveric experiment.

STUDY DESIGN: A feasibility study with three-dimensional surgical simulation and cadaveric experiment. OBJECTIVE: To verify the feasibility of a new navigated surgical method for lumbosacral interbody fusion. SUMMARY OF BACKGROUND DATA: The advances in surgical navigation have opened new possibilities for lumbosacral interbody fusion procedure. We designed a novel navigated surgical method that enables lumbosacral discectomy and bone grafting to be performed percutaneously and safely. METHODS: First, to prove that the newly designed surgical method is feasible from an anatomic perspective, the new method, navigated percutaneous lumbosacral interbody fusion (NPLSIF), was simulated on the three-dimensional models of lumbosacral spine. The three-dimensional models were established using the computed tomographic (CT) data of 60 patients. Feasibility could be verified if both working corridor and S1 pedicle screw could be accommodated in sacral ala without overlapping and without penetrating either the spinal canal or the anterior or upper sacral wall. Second, to verify the feasibility of the NPLSIF procedure in reality, cadaveric experiment was performed. Two cadavers were included; one was a 67-year-old male, and the other a 65-year-old female. CT scanning was performed with an intraoperative CT scanner before surgery, after the discectomy and after bone grafting. These three series of CT images were compared to evaluate the efficacy of the NPLSIF procedure. After the procedures, the lumbosacral spines were separated from the cadaver trunks in the department of anatomy. The lumbosacral disc of one cadaver was bisected coronally, while the lumbosacral disc of the other cadaver was bisected sagittally. The internal view of the lumbosacral discs helped to further evaluate the efficacy of the NPLSIF procedures. RESULTS: In the three-dimensional surgical simulation experiment, the feasibility of the NPLSIF procedure was verified in every case. In the cadaveric experiment, the NPLSIF procedures were successfully executed. The surgical procedure on the first cadaver took 3 hours. After improving the workflow and having gained some experience, the procedure on the second cadaver took 1.5 hours. On the navigation workstation, the preoperative plan was completed in 3 to 5 minutes and each intraoperative CT scanning took 30 seconds. The quality of the intraoperative CT images was comparable to that of normal CT images. CT images and the internal view of the lumbosacral discs showed that the NPLSIF procedures had yielded satisfactory discectomy and endplate preparation. CONCLUSION: The feasibility of NPLSIF was verified by the means of three-dimensional surgical simulation and cadaveric experiment. Clinical studies are needed to further investigate the efficacy and efficiency of NPLSIF in clinical practice.

Navigated percutaneous lumbosacral interbody fusion: a feasibility study.

INTRODUCTION: Advances in surgical navigation have opened new possibilities for lumbosacral interbody fusion procedures. We have designed a novel navigated surgical method, Navigated Percutaneous Lumbosacral Interbody Fusion (NPLSIF), that enables lumbosacral discectomy and bone grafting to be performed percutaneously and safely. METHODS: To prove that NPLSIF is feasible from an anatomical perspective, it was simulated on 3D models of the lumbosacral spine created using CT data from 60 patients. Feasibility would be verified if both the working corridor and the S1 pedicle screw could be accommodated in the sacral ala without overlapping and without penetrating either the spinal canal or the anterior or upper sacral wall. In addition, the discectomy that could be achieved using NPLSIF was evaluated, and a surgical experiment was performed using a plastic torso model. RESULTS: The 3D modeling and surgical simulation were successfully completed in all cases. The feasibility of the NPLSIF approach was verified in every case, i.e., both the working corridor and the S1 pedicle screw were accommodated in the sacral ala without overlapping and without penetrating either the spinal canal or the anterior or upper sacral wall. The mean ratio of the area of discectomy that could be achieved by NPLSIF to the total area of the lumbosacral disc was 0.721 ± 0.065 (range: 0.57-0.894), 0.956 ± 0.045 (range: 0.8-1.0) and 0.945 ± 0.058 (range: 0.813-1.0) in the axial, coronal and sagittal planes, respectively. NPLSIF was also successfully executed on the plastic torso model. Preoperative planning on the navigation workstation took 5 minutes and each intraoperative CT scan took 30 seconds. Locating the entry point of the working corridor according to the preoperative plan took 3 minutes. Postoperative CT images and direct viewing of the plastic model revealed no penetration of the spinal canal or sacral wall. CONCLUSION: The feasibility of Navigated Percutaneous Lumbosacral Interbody Fusion (NPLSIF) was verified from an anatomical perspective. We also demonstrated that an adequate discectomy can be achieved using the procedure. Cadaveric experiments and clinical trials are needed to further evaluate the efficacy and efficiency of NPLSIF.

Self-assembled composite matrix in a hierarchical 3-D scaffold for bone tissue engineering.

It is of high clinical relevance in bone tissue engineering that scaffolds promote a high seeding efficiency of cells capable of osteogenic differentiation, such as human bone marrow-derived mesenchymal stem cells (hMSCs). We evaluated the effects of a novel polycaprolactone (PCL) scaffold on hMSC seeding efficiency, proliferation, distribution and differentiation. Porous PCL meshes prepared by fused deposition modeling (FDM) were embedded in matrix of hyaluronic acid, methylated collagen and terpolymer via polyelectrolyte complex coacervation. Scaffolds were cultured statically and dynamically in osteogenic stimulation medium for up to 28 days. Compared to naked PCL scaffolds, embedded scaffolds provided a higher cell seeding efficiency (t-test, P<0.05), a more homogeneous cell distribution and more osteogenically differentiated cells, verified by a more pronounced gene expression of the bone markers alkaline phosphatase, osteocalcin, bone sialoprotein I and bone sialoprotein II. Dynamic culture resulted in higher amounts of DNA (day 14 and day 21) and calcium (day 21 and day 28), compared to static culture. Dynamic culture and the embedding synergistically enhanced the calcium deposition of hMSC on day 21 and day 28. This in vitro study provides evidence that hybrid scaffolds made from natural and synthetic polymers improve cellular seeding efficiency, proliferation, distribution and osteogenic differentiation.