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Background Context: Interbody spinal fusion relies on the use of external fixation and the placement of a fusion cage filled with graft materials (scaffolds) without regard for their mechanical performance. Stability at the fusion site is instead reliant on fixation hardware combined with a selected cage. Ideally, scaffolds placed into the cage should both support the formation of new bone and contribute to the mechanical stability at the fusion site., Purpose: We recently developed a scaffold consisting of silane-modified PCL-TCP (PCL-siTCP) with mechanical properties that can withstand the higher loads generated in the spine. To ensure the scaffold more closely mimicked the bone matrix, we incorporated collagen (Col) and a heparan sulfate glycosaminoglycan sugar (HS3) with increased affinity for heparin-binding proteins such as bone morphogenetic protein-2 (BMP-2). The osteostimulatory characteristic of this novel device delivering exogenous BMP2 was assessed in vitro and in vivo as a prelude to future spinal fusion studies with this device., Study Design/setting: A combination of cell-free assays (BMP2 release), progenitor cell-based assays (BMP2 bioactivity, cell proliferation and differentiation), and rodent ectopic bone formation assays was used to assess the osteostimulatory characteristics of the PCL-siTCP-based scaffolds., Materials and Methods: Freshly prepared rat mesenchymal stem cells were used to determine reparative cell proliferation and differentiation on the PCL-siTCP-based scaffolds over a 28-day period in vitro. The bioactivity of BMP2 released from the scaffolds was assessed on progenitor cells over a 28-day period using ALP activity assays and release kinetics as determined by enzyme-linked immunosorbent assay. For ectopic bone formation, intramuscular placement of scaffolds into Sprague Dawley rats (female, 4 weeks old, 120-150 g) was achieved in five animals, each receiving four treatments randomized for location along the limb. The four groups tested were (1) PCL-siTCP/Col (5-mm diameter×1-mm thickness), PCL-siTCP/Col/BMP2 (5 µg), (3) PCL-siTCP/Col/HS3 (25 µg), and (4) PCL-siTCP/Col/HS3/BMP2 (25 and 5 µg, respectively). Bone formation was evaluated at 8 weeks post implantation by microcomputed tomography (µCT) and histology., Results: Progenitor cell-based assays (proliferation, mRNA transcripts, and ALP activity) confirmed that BMP2 released from PCL-siTCP/Col/HS3 scaffolds increased ALP expression and mRNA levels of the osteogenic biomarkers Runx2, Col1a2, ALP, and bone gla protein-osteocalcin compared with devices without HS3. When the PCL-siTCP/Col/HS3/BMP2 scaffolds were implanted into rat hamstring muscle, increased bone formation (as determined by two-dimensional and three-dimensional µCTs and histologic analyses) was observed compared with scaffolds lacking BMP2. More consistent increases in the amount of ectopic bone were observed for the PCL-siTCP/Col/HS3/BMP2 implants compared with PCL-siTCP/Col/BMP2. Also, increased mineralizing tissue within the pores of the scaffold was seen with modified-tetrachrome histology, a result confirmed by µCT, and a modest but detectable increase in both the number and the thickness of ectopic bone structures were observed with the PCL-siTCP/Col/HS3/BMP2 implants., Conclusions: The combination of PCL-siTCP/Col/HS3/BMP2 thus represents a promising avenue for further development as a bone graft alternative for spinal fusion surgery., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The Wharton's Jelly (WJ) is an established source of mesenchymal stem cells (MSC). We compared 3 methods of extracting WJ-MSC from cryopreserved tissue and determined that enzymatic digestion of the WJ yielded the most viable MSC, compared to the explant and mechanical digestion methods. The enzymatically-released WJ-MSC conformed to the International Society for Cellular Therapy (ISCT) criteria: displayed plastic-adherence, co-expressed CD73, CD90, CD105 and were negative for hematopoietic lineage cell markers.

In the present study, polycaprolactone-tricalcium phosphate (PCL/TCP) scaffolds with two different fibre laydown patterns, which were coated with hydroxyapatite and gelatine, were used as an approach for optimizing bone regeneration in a critical-sized calvarial defect. After 12 weeks, bone regeneration was quantified using microcomputed tomography (micro-CT) analysis, biomechanical testing, and histological evaluation. Notably, the experimental groups with coated scaffolds showed lower bone formation and lower biomechanical properties within the defect compared to the uncoated scaffolds. Surprisingly, the different laydown pattern of the fibres resulted in different bone formation and biomechanical properties: the 0°/60°/120° scaffolds revealed lower bone formation and biomechanical properties compared to the 0°/90° scaffolds in all the experimental groups. Therefore, future bone regeneration strategies utilizing scaffolds should consider scaffold architecture as an important factor during the scaffold optimization stages in order to move closer to a clinical application., (Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.)

A significant stumbling block in the creation of functional three-dimensional (3D) engineered tissues is the proper vascularization of the constructs. Furthermore, in the context of electrospinning, the development of 3D constructs using this technique has been hindered by the limited infiltration of cells into their structure. In an attempt to address these issues, a hybrid mesh of poly (ɛ-caprolactone)-collagen blend (PCL/Col) and hyaluronic acid (HA) hydrogel, Heprasil™ was created via a dual electrodeposition system. Simultaneous deposition of HA and PCL/Col allowed the dual loading and controlled release of two potent angiogenic growth factors VEGF(165) and PDGF-BB over a period of five weeks in vitro. Furthermore, this manner of loading sustained the bioactivity of the two growth factors. Utilizing an in-house developed 3D co-culture assay model of human umbilical vein endothelial cells and lung fibroblasts, the growth factor-loaded hybrid meshes was shown to not only support cellular attachment, but also their infiltration and the recapitulation of primitive capillary network in the scaffold's architecture. Thus, the creation of a PCL/Col-Heprasil hybrid scaffold is a step forward toward the attainment of a 3D bio-functionalized, vascularized tissue engineering construct., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The objective of this present study was to improve the functional performance of rapid prototyped scaffolds for bone tissue engineering through biomimetic composite coating. Rapid prototyped poly(ε-caprolactone)/tri-calcium phosphate (PCL/TCP) scaffolds were fabricated using the screw extrusion system (SES). The fabricated PCL/TCP scaffolds were coated with a carbonated hydroxyapatite (CHA)-gelatin composite via biomimetic co-precipitation. The structure of the prepared CHA-gelatin composite coating was studied by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. Compressive mechanical testing revealed that the coating process did not have any detrimental effect on the mechanical properties of the scaffolds. The cell-scaffold interaction was studied by culturing porcine bone marrow stromal cells (BMSCs) on the scaffolds and assessing the proliferation and bone-related gene and protein expression capabilities of the cells. Confocal laser microscopy and SEM images of the cell-scaffold constructs showed a uniformly distributed cell sheet and accumulation of extracellular matrix in the interior of CHA-gelatin composite-coated PCL/TCP scaffolds. The proliferation rate of BMSCs on CHA-gelatin composite-coated PCL/TCP scaffolds was about 2.3 and 1.7 times higher than that on PCL/TCP scaffolds and CHA-coated PCL/TCP scaffolds, respectively, by day 10. Furthermore, reverse transcription polymerase chain reaction and Western blot analysis revealed that CHA-gelatin composite-coated PCL/TCP scaffolds stimulate osteogenic differentiation of BMSCs the most, compared with PCL/TCP scaffolds and CHA-coated PCL/TCP scaffolds. These results demonstrate that CHA-gelatin composite-coated rapid prototyped PCL/TCP scaffolds are promising for bone tissue engineering., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

Conventional clinical therapies are unable to resolve osteochondral defects adequately; hence, tissue engineering solutions are sought to address the challenge. A biphasic implant that was seeded with mesenchymal stem cells (MSCs) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a polycaprolactone (PCL) cartilage scaffold and a PCL-tricalcium phosphate osseous matrix. Autologous MSCs were seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL-collagen electrospun mesh, which served as a substitute for periosteal flap in preventing cell leakage. Controls without either implanted MSCs or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by superior glycosaminoglycan maintenance. This positive morphological outcome was supported by a higher relative Young's modulus, which indicated functional cartilage restoration. Bone ingrowth and remodeling occurred in all groups, with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover, healing was inferior at the patellar groove when compared with the medial condyle and this was attributed to the native biomechanical features.

In this study, poly (epsilon-caprolactone) [PCL] and its collagen composite blend (PCL/Col) were fabricated to scaffolds using electrospinning method. Incorporated collagen was present on the surface of the fibers, and it modulated the attachment and proliferation of pig bone marrow mesenchymal cells (pBMMCs). Osteogenic differentiation markers were more pronounced when these cells were cultured on PCL/Col fibrous meshes, as determined by immunohistochemistry for collagen type I, osteopontin, and osteocalcin. Matrix mineralization was observed only on osteogenically induced PCL/Col constructs. Long bone analogs were created by wrapping osteogenic cell sheets around the PCL/Col meshes to form hollow cylindrical cell-scaffold constructs. Culturing these constructs under dynamic conditions enhanced bone-like tissue formation and mechanical strength. We conclude that electrospun PCL/Col mesh is a promising material for bone engineering applications. Its combination with osteogenic cell sheets offers a novel and promising strategy for engineering of tubular bone analogs.

A common problem in the design of tissue engineered scaffolds using electrospun scaffolds is the poor cellular infiltration into the structure. To tackle this issue, three approaches to scaffold design using electrospinning were investigated: selective leaching of a water-soluble fiber phase (poly ethylene oxide (PEO) or gelatin), the use of micron-sized fibers as the scaffold, and a combination of micron-sized fibers with codeposition of a hyaluronic acid-derivative hydrogel, Heprasil. These designs were achieved by modifying a conventional electrospinning system with two charged capillaries and a rotating mandrel collector. Three types of scaffolds were fabricated: medical grade poly(epsilon-caprolactone)/collagen (mPCL/Col) cospun with PEO or gelatin, mPCL/Col meshes with micron-sized fibers, and mPCL/Col microfibers cosprayed with Heprasil. All three scaffold types supported attachment and proliferation of human fetal osteoblasts. However, selective leaching only marginally improved cellular infiltration when compared to meshes obtained by conventional electrospinning. Better cell penetration was seen in mPCL/Col microfibers, and this effect was more pronounced when Heprasil regions were present in the structure. Thus, such techniques could be further exploited for the design of cell permeable fibrous meshes for tissue engineering applications.

Scaffolds manufactured from biological materials promise better clinical functionality, providing that characteristic features are preserved. Collagen, a prominent biopolymer, is used extensively for tissue engineering applications, because its signature biological and physico-chemical properties are retained in in vitro preparations. We show here for the first time that the very properties that have established collagen as the leading natural biomaterial are lost when it is electro-spun into nano-fibres out of fluoroalcohols such as 1,1,1,3,3,3-hexafluoro-2-propanol or 2,2,2-trifluoroethanol. We further identify the use of fluoroalcohols as the major culprit in the process. The resultant nano-scaffolds lack the unique ultra-structural axial periodicity that confirms quarter-staggered supramolecular assemblies and the capacity to generate second harmonic signals, representing the typical crystalline triple-helical structure. They were also characterised by low denaturation temperatures, similar to those obtained from gelatin preparations (p>0.05). Likewise, circular dichroism spectra revealed extensive denaturation of the electro-spun collagen. Using pepsin digestion in combination with quantitative SDS-PAGE, we corroborate great losses of up to 99% of triple-helical collagen. In conclusion, electro-spinning of collagen out of fluoroalcohols effectively denatures this biopolymer, and thus appears to defeat its purpose, namely to create biomimetic scaffolds emulating the collagen structure and function of the extracellular matrix.

In this study, a nanofiber mesh made by co-electrospinning medical grade poly(epsilon-caprolactone) and collagen (mPCL/Col) was fabricated and studied. Its mechanical properties and characteristics were analyzed and compared to mPCL meshes. mPCL/Col meshes showed a reduction in strength but an increase in ductility when compared to PCL meshes. In vitro assays revealed that mPCL/Col supported the attachment and proliferation of smooth muscle cells on both sides of the mesh. In vivo studies in the corpus cavernosa of rabbits revealed that the mPCL/Col scaffold used in conjunction with autologous smooth muscle cells resulted in better integration with host tissue when compared to cell free scaffolds. On a cellular level preseeded scaffolds showed a minimized foreign body reaction.

Current clinical therapies for traumatic or chronic injuries involving osteochondral tissue result in temporary pain reduction and filling of the defect but with biomechanically inferior repair tissue. Tissue engineering of osteochondral repair tissue using autologous cells and bioactive biomaterials has the potential to overcome the current limitations and results in native-like repair tissue with good integration capabilities. For this reason, we applied two modem biomaterial design techniques, namely, electrospinning and fused deposition modeling (FDM), to produce bioactive poly(epsilon-caprolactone)/collagen (PCL/Col) type I and type II-PCL-tri-calcium phosphate (TCP)/Col composites for precursor cell-based osteochondral repair. The application of these two design techniques (electrospinning and FDM) allowed us to specifically produce the a suitable three-dimensional (3D) environment for the cells to grow into a particular tissue (cartilage and bone) in vitro prior to in vivo implantation. We hypothesize that our new designed biomaterials, seeded with autologous bone marrow-derived precursor cells, in combination with bioreactor-stimulated cell-culture techniques can be used to produce clinically relevant osteochondral repair tissue.