Granulation tissue formation requires a robust angiogenic response. As granulation tissue develops, collagen fibers are deposited and compacted. Forces generated in the wake of this process drive wound contraction to reduce the wound area. In diabetics, both angiogenesis and wound contraction are diminished leading to impaired wound healing. To emulate this pathology and to address it pharmacologically, we developed a wound healing model in the diabetic Zucker fatty rat and tested a topical proangiogenic strategy combining antifungal agent ciclopirox olamine (CPX) and lysophospholipid sphingosine-1-phosphate (S1P) to promote diabetic wound closure. In vitro, we demonstrated that CPX + S1P up-regulates a crucial driver of angiogenesis, hypoxia-inducible factor-1, in endothelial cells. Injection of CPX + S1P into subcutaneously implanted sponges in experimental rats showed, in an additive manner, a fivefold increased endothelial infiltration and lectin-perfused vessel length. We developed a splinted diabetic rodent model to achieve low wound contraction rates that are characteristic for the healing mode of diabetic ulcers in humans. We discovered specific dorsal sites that allowed for incremental full-thickness excisional wound depths from 1 mm (superficial) to 3 mm (deep). This enabled us to bring down wound contraction from 51% in superficial wounds to 8% in deep wounds. While the effects of topical gel treatment of CPX + S1P were masked by the rodent-characteristic dominant contraction in superficial wounds, they became clearly evident in deep diabetic wounds. Here, a fivefold increase of functional large vessels resulted in accelerated granulation tissue formulation, accompanied by a 40% increase of compacted thick collagen fibers. This was associated with substantially reduced matrix metalloproteinase-3 and -13 expression. These findings translated into a fivefold increase in granulation-driven contraction, promoting diabetic wound closure. With CPX and S1P analogues already in clinical use, their combination presents itself as an attractive proangiogenic treatment to be repurposed for diabetic wound healing.

Platelet-rich plasma (PRP) is a volume of autologous plasma that has a higher platelet concentration above baseline. It has already been approved as a new therapeutic modality and investigated in clinics, such as bone repair and regeneration, and oral surgery, with low cost-effectiveness ratio. At present, PRP is mostly prepared using a centrifuge. However, this method has several shortcomings, such as long preparation time (30 min), complexity in operation, and contamination of red blood cells (RBCs). In this paper, a new PRP preparation approach was proposed and tested. Ultrasound waves (4.5 MHz) generated from piezoelectric ceramics can establish standing waves inside a syringe filled with the whole blood. Subsequently, RBCs would accumulate at the locations of pressure nodes in response to acoustic radiation force, and the formed clusters would have a high speed of sedimentation. It is found that the PRP prepared by the proposed device can achieve higher platelet concentration and less RBCs contamination than a commercial centrifugal device, but similar growth factor (i.e., PDGF-ββ). In addition, the sedimentation process under centrifugation and sonication was simulated using the Mason-Weaver equation and compared with each other to illustrate the differences between these two technologies and to optimize the design in the future. Altogether, ultrasound method is an effective method of PRP preparation with comparable outcomes as the commercially available centrifugal products.

Blend emulsion electrospinning is widely perceived to destroy the bioactivity of proteins, and a blend emulsion of water-soluble and nonsoluble molecules is believed to be thermodynamically unstable to electrospin smoothly. Here we demonstrate a method to retain the bioactivity of disparate fragile biomolecules when electrospun. Using bovine serum albumin as a carrier protein; water-soluble vitamin C, fat soluble vitamin D3, steroid hormone hydrocortisone, peptide hormone insulin, thyroid hormone triiodothyronine (T3), and peptide epidermal growth factor (EGF) were simultaneously blend-spun into PLGA-collagen nanofibers. Upon release, vitamin C maintained the ability to facilitate Type I collagen secretion by fibroblasts, EGF stimulated skin fibroblast proliferation, and insulin potentiated adipogenic differentiation. Transgenic cell reporter assays confirmed the bioactivity of vitamin D3, T3, and hydrocortisone. These factors concertedly increased keratinocyte and fibroblast proliferation while maintaining keratinocyte basal state. This method presents an elegant solution to simultaneously deliver disparate bioactive biomolecules for wound healing applications.

This study investigated the adhesive behaviors of normal and abnormal hematopoietic cells on nanotopographical materials. Previously, electrospun nanofiber scaffolds (NFSs) were used to capture and expand hematopoietic stem cells in vitro; here, we demonstrate that NFS could also serve as a useful bioadhesive platform for capturing functionally adherent leukemia cells. Collagen-blended poly(d,l-lactide-co-glycolide) NFS enabled more rapid and efficient capture of K562 leukemia cells than tissue culture polystyrene surfaces with up to 70% improved adhesion and shorter time. Cellular extensions, stronger adhesion, and enhanced cell-cell interactions were observed in K562 cells captured on NFS. While NFS promoted hematopoietic progenitor cell proliferation, it inhibited leukemia cell proliferation and affected cell cycle status by shifting more cells toward the G0/G1 phase. The expression of α-integrins was equally high in both captured and uncaptured leukemia cell populations demonstrating no relation to its adhesive nature. Hematopoietic morphological signatures of NFS captured cells presented no impact on cell differentiation. We conclude that electrospun NFS serves as an excellent platform not only for capturing functionally adherent leukemia cells but also for studying the impact of niche-like structure in the nanoscale.

The design of biomimetic nanomaterials that can directly influence the behavior of cells and facilitate the regeneration of tissues and organs has become an active area of research. Here, the production of materials based on nano‐hydroxyapatite composites in scaffolds with nanofibrous and nanoporous topographies, designed to mimic the native bone matrix for applications in bone tissue engineering, is reported. Human mesenchymal stem cells grown on these nanocomposites are stimulated to rapidly produce bone minerals in situ, even in the absence of osteogenic supplements in the cell‐culture medium. Nanocomposites comprising type I collagen and nano‐hydroxyapatite are found to be especially efficient at inducing mineralization. When subcutaneously implanted into nude mice, this biomimetic nanocomposite is able to form a new bone matrix within only two weeks. Furthermore, when the nanocomposite is enriched with human mesenchymal stem cells before implantation, development of the bone matrix is accelerated to within one week. To the best of the authors' knowledge, this study provides the first clear in vitro and in vivo demonstration of osteoinduction controlled by the material characteristics of a biomimetic nanocomposite. This approach can potentially facilitate the translation of de novo bone‐formation technologies to the clinic.

Dexamethasone (Dex), a synthetic corticosteroid, was loaded into poly(L-lactic acid) (PLLA) nanofibrous scaffolds with a concentration of 0.333 wt% by electrospinning. The Dex-loaded PLLA nanofibres increased the mechanical strength in comparison with pure PLLA nanofibres. A sustained release profile for over 2 months with an initial burst release after 12 h of 17% was shown. Importantly, the amounts of Dex released from the PLLA nanofibres every 3 days were close to the ones used for the standard osteogenic medium. The sustained osteoinductive environment created by released Dex strongly differentiated human mesenchymal stem cells (hMSCs) cultured in the Ost(-Dex) medium. ALP activity, BSP expression and calcium deposition were significantly higher than those of the cells cultured on the PLLA scaffolds without Dex. A large amount of hydroxyapatite-like minerals was observed on the Dex-loaded PLLA scaffolds after 21 days culture. The cells on these scaffolds also indicated an osteoblastic morphology on the 14th day. Besides, these scaffolds slightly increased the cell proliferation comparing to the scaffolds without Dex. As such, the PLLA nanofibres loaded with 0.333 wt% Dex was an effective osteoinductive scaffold which acts as a promising strategy for bone treatment.

Aim: Developing 3D scaffolds mimicking the nanoscale structure of the native extracellular matrix is important in tissue regeneration. In this study, we aimed to demonstrate the novelty of 3D nanofibrous scaffolds and compare their efficiency with 2D nanofibrous scaffolds. Materials & methods: The 2D poly(L-lactic acid)/collagen nanofibrous scaffolds were 2D meshes fabricated by the conventional electrospinning technique, whereas the 3D poly(L-lactic acid)/collagen nanofibrous scaffolds were fabricated by a modified electrospinning technique using a dynamic liquid support system. The morphology, proliferation and differentiation abilities of human mesenchymal stem cells in osteogenic medium on both scaffolds were investigated. Results & conclusion: Compared with the 2D scaffolds, the 3D scaffolds significantly increased the expression of osteoblastic genes of the stem cells as well as the formation of bone minerals. In addition, the scanning electron microscopic and micro-computed tomographic images showed the dense deposition of bone minerals aligned along the nanofibers of the 3D scaffolds after 14 and 28 days cultured with the mesenchymal stem cells. As such, the 3D electrospun poly(L-lactic acid)/collagen nanofibrous scaffold is a novel bone graft substitute for bone tissue regeneration. Original submitted 17 November 2011; Revised submitted 21 February 2012; Published online 18 June 2012

Extracellular matrix (ECM) of bone tissue is a typical composite system consisting of a mineral phase (hydroxyapatite [HA]—more precisely bone mineral is a hydroxyl-and calcium-decient, carbonated apatite) and an organic phase (collagen and noncollagenous proteins such as osteocalcin, osteonectin, bone sialoprotein) (Boskey 2005). This composite ECM structure can be described in terms of hierarchical levels of organization from nanometers, through micrometers, and nally to millimeters scale range (Rho et al. 1998).

Using scaffolds with autologous stem cells is a golden strategy for the treatment of bone defects. In this strategy, human mesenchymal stem cells (hMSCs) have often been isolated and expanded in vitro on a plastic surface to obtain a sufficient cell number before seeding on a suitable scaffold. Materials & Methods: Investigating the influence of serial passages (from passage two to passage eight) on the abilities of proliferation and osteogenic differentiation of hMSCs on 24-well tissue culture polystyrene plates and poly l-lactic acid electrospun nanofibrous scaffolds was performed to determine how prolonged culture affected these cellular abilities and how the nanofibrous scaffolds supported the osteogenic differentiation potential of hMSCs. Results & Conclusion: Serial passage caused adverse changes in hMSCs characteristics, which were indicated by the decline in both proliferation and osteogenic differentiation abilities. Interestingly, the poly l-lactic acid nanofibrous scaffolds showed a significant support in recovering the osteogenic abilities of hMSCs, which had been severely affected by prolonged culture.

Mesenchymal Stromal Cell (MSC) co-culture enhances ex vivo expansion of hematopoietic stem and progenitor cells from thawed Umbilical Cord Blood (UCB) without prior CD34+/CD133+ cell selection. We hypothesized that the superior ex vivo expansion is due to reversal of UCB c apoptosis via intercellular cytosolic transfer from the MSC to the UCB cells. MSC endogenously expressing green fluorescent protein (GFP) or exogenously labeled with quantum dots (QDs) were co-cultured with UCB cells positive for a hematopoietic-specific CD45 marker. Cultured UCB cells without MSC layer served as control. Annexin-V, JC-1 dye & caspase inhibitors were used to investigate mitochondria-associated apoptosis pathway. Flow cytometry & confocal imaging were used to detect for the transfer of the GFP or QD in co-cultured UCB cells. Thawed UCB cells had high percentage of early apoptotic cells (defined by Annexin-V positivity) which progressed to dead cells (defined by 7AAD positivity). MSC co-culture resulted in reversal of Annexin-V positivity and reached a maximum on day 3 (viability defined by double negative staining for Annexin-V and 7AAD) (p < 0.05). A significant reduction in caspase 3/7, 8 & 9 activity as well as preservation of mitochondrial membrane potential were observed in the co-cultured UCB cells (p < 0.05). Upon co-culture with GFP-expressing or QD-labelled MSC, unlabelled UCB cells were found positive for these markers as early as 24 hours post co-culture. The number of GFP & CD45 positive UCB cells increased over the first 3 days of co-culturing, which correlated with viability increase. Direct contact between MSC & UCB was necessary for the transfer, as negligible transfer was observed when the two populations were separated by transwell inserts. Separation also showed a significant decrease in viability. The UCB cells that tightly adhered to the MSC layer exhibited optimal transfer & cell viability. DNA analysis of the viable, GFP transfer-positive UCB cells, as determined by Variable Number Tandem Repeat analysis ruled out the possibilities of MSC trans-differentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB cell viability (p < 0.0001). The above data provides evidence that intercellular transfer of cytosolic materials could be one novel mechanism preventing UCB cell death in MSC co-culture. Mesenchymal Stromal Cell (MSC) co-culture enhances ex vivo expansion of hematopoietic stem and progenitor cells from thawed Umbilical Cord Blood (UCB) without prior CD34+/CD133+ cell selection. We hypothesized that the superior ex vivo expansion is due to reversal of UCB c apoptosis via intercellular cytosolic transfer from the MSC to the UCB cells. MSC endogenously expressing green fluorescent protein (GFP) or exogenously labeled with quantum dots (QDs) were co-cultured with UCB cells positive for a hematopoietic-specific CD45 marker. Cultured UCB cells without MSC layer served as control. Annexin-V, JC-1 dye & caspase inhibitors were used to investigate mitochondria-associated apoptosis pathway. Flow cytometry & confocal imaging were used to detect for the transfer of the GFP or QD in co-cultured UCB cells. Thawed UCB cells had high percentage of early apoptotic cells (defined by Annexin-V positivity) which progressed to dead cells (defined by 7AAD positivity). MSC co-culture resulted in reversal of Annexin-V positivity and reached a maximum on day 3 (viability defined by double negative staining for Annexin-V and 7AAD) (p < 0.05). A significant reduction in caspase 3/7, 8 & 9 activity as well as preservation of mitochondrial membrane potential were observed in the co-cultured UCB cells (p < 0.05). Upon co-culture with GFP-expressing or QD-labelled MSC, unlabelled UCB cells were found positive for these markers as early as 24 hours post co-culture. The number of GFP & CD45 positive UCB cells increased over the first 3 days of co-culturing, which correlated with viability increase. Direct contact between MSC & UCB was necessary for the transfer, as negligible transfer was observed when the two populations were separated by transwell inserts. Separation also showed a significant decrease in viability. The UCB cells that tightly adhered to the MSC layer exhibited optimal transfer & cell viability. DNA analysis of the viable, GFP transfer-positive UCB cells, as determined by Variable Number Tandem Repeat analysis ruled out the possibilities of MSC trans-differentiation or MSC-UCB fusion. In addition, there was statistical correlation between higher levels of cytosolic transfer and enhanced UCB cell viability (p < 0.0001). The above data provides evidence that intercellular transfer of cytosolic materials could be one novel mechanism preventing UCB cell death in MSC co-culture.

Nanostructured materials are gaining new impetus owing to the advancements in material fabrication techniques and their unique properties (their nanosize, high surface area-to-volume ratio, and high porosity). Such nanostructured materials mimic the subtleties of extracellular matrix (ECM) proteins, creating artifi cial microenvironments which resemble the native niches in the body. On the other hand, the isolation of mesenchymal stem cells (MSCs) from various tissue sources has resulted in the interest to study the multiple differentiation lineages for various therapeutic treatments. In this review, our focus is tailored towards the potential of biomimetic nanostructured materials as osteoinductive scaffolds for bone regeneration to differentiate MSCs towards osteoblastic cell types without the presence of soluble factors. In addition to mimicking the nanostructure of native bone, the supplement of collagen and hydroxyapatite which mimic the main components of the ECM also brings signifi cant advantages to these materials.

Acute full-thickness skin wounds (FTSW) caused by extensive burns or high-energy trauma are not adequately addressed by current clinical treatments. This study hypothesized that biomimetic nanofiber scaffolds (NFSs) functionalized with rich attachment of bone-marrow-derived mesenchymal stem cells (BM-MSCs) can promote wound healing in acute FTSW. Results in a rat model showed that both NFS and BM-MSCs contributed to the wound healing. Wounds in NFS group with a higher density of BM-MSCs achieved complete closure 8 days earlier than the control group. Implanted BM-MSCs were found to promote epithelial edge ingrowth and collagen synthesis. The colocation of BM-MSCs (tagged with quantum-dots) with the expression of keratin 10 and filaggrin indicated the participation of BM-MSCs in epidermal differentiation at early and intermediate stages under the local wounding environment. Overall, this study suggests a great potential of using NFS/BM-MSC composites for the treatment of acute FTSW.

We evaluate the feasibility of applying polarized Raman spectroscopy in probing the early biochemical compositions and orientation changes in impacted porcine cartilage explants. We divide 100 fresh tibial cartilage explants into four groups: control (unimpacted) and 3 groups of single impact at 15, 20, and 25 MPa. Each group is examined for biochemical changes using Raman microscopy, cell viability changes using confocal fluorescence microscopy, and histological changes using the modified Mankin score. For the 15-MPa impact group, the modified Mankin score (p>0.05, n=15) and cell viability test (p>0.05, n=5) reveal no significant changes when compared to the control, but polarized Raman spectroscopy detects significant biochemical changes. A significant decrease in the parallel polarized intensity of the pyranose ring band at 1126 cm(-1) suggests a possible decrease in the glycoaminoglycan content in early cartilage damage (one-way analysis of variance with a post hoc Bonferonni test, p<0.05, n=10). For impacts greater than 15 MPa, cell viability and modified Mankin score are consistent with the changes in the observed polarized Raman signals. This suggests that the polarized Raman spectroscopy technique has potential for diagnosis and detection of early cartilage damage at the molecular level.

Mechanical stimulation is one of the factors that regulating bone regeneration and healing. In this study, the biological responses of bone marrow derived mesenchymal stem cells (MSCs) to mechanical stimuli on aligned nanofibers and cast films were investigated. The uniaxial cyclic strain (1% strain and 1 Hz) was applied continuously to the cell substrates and osteoblastic activities were assessed at weeks 1, 2, and 4. The MSCs morphology on the aligned nanofibers was more elongated and spindle-like than MSCs on the cast films. Strain stimulation significantly attenuated the proliferation at week one but was significantly enhanced at week 4 for both types of substrates. Only the MSCs on strained nanofibers had greater alkaline phosphatase (ALP) levels at week one, while the ALP hindered the MSCs on both substrates at week 4. Strain application played a greater influence on osteocalcin expression for the cast films than the nanofibers at week 4. Clearly, the cellular response to strain induction was highly dependent on the surface-cell adhesion, which itself was greatly influenced by the surface texture of the substrate.

The time required for osseointegration with a metal implant having a smooth surface ranges from three to six months. We hypothesized that biomimetic coating surfaces with poly(lactic-co-glycolic acid) (PLGA)/collagen fibers and nano-hydroxyapatite (n-HA) on the implant would enhance the adhesion of mesenchymal stem cells. Therefore, this surface modification of dental and bone implants might enhance the process of osseointegration. In this study, we coated PLGA or PLGA/collagen (50:50 w/w ratio) fiber on Ti disks by modified electrospinning for 5 s to 2 min; after that, we further deposited n-HA on the fibers. PLGA fibers of fiber diameter 0.957 ± 0.357 µm had a contact angle of 9.9 ± 0.3° and PLGA/collagen fibers of fiber diameter 0.378 ± 0.068 µm had a contact angle of 0°. Upon n-HA incorporation, all the fibers had a contact angle of 0° owing to the hydrophilic nature of n-HA biomolecule. The cell attachment efficiency was tested on all the scaffolds for different intervals of time (10, 20, 30 and 60 min). The alkaline phosphatase activity, cell proliferation and mineralization were analyzed on all the implant surfaces on days 7, 14 and 21. Results of the cell adhesion study indicated that the cell adhesion was maximum on the implant surface coated with PLGA/collagen fibers deposited with n-HA compared to the other scaffolds. Within a short span of 60 min, 75% of the cells adhered onto the mineralized PLGA/collagen fibers. Similarly by day 21, the rate of cell proliferation was significantly higher (p ⩽ 0.05) on the mineralized PLGA/collagen fibers owing to enhanced cell adhesion on these fibers. This enhanced initial cell adhesion favored higher cell proliferation, differentiation and mineralization on the implant surface coated with mineralized PLGA/collagen fibers.