Directional Cell Migration Guide for Improved Tissue Regeneration.

The field of tissue regeneration has seen a paradigm shift after one wave of technological innovation after another, which has notably made significant contributions to basic cellular response control and overall tissue regeneration. One particular area that is seeing rekindled interest after technological innovation is managing cell migration toward defects because successful host cell migration from adjacent tissue can accelerate overall regeneration time in tissue defects that are either large in size or irregular in shape. This chapter surveys significant advances on directed cell migration upon topological cues. First, we introduce several examples of patterning and electrospinning technology for guiding directed cell migration, followed by a discussion on approaches to influencing radially aligned topography in pattern or electrospun sheet for overall tissue regeneration.

Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects.

Over the last few decades, stem cell therapies have been highlighted for their potential to heal damaged tissue and aid in tissue reconstruction. However, materials used to deliver and support implanted cells often display limited efficacy, which has resulted in delaying translation of stem cell therapies into the clinic. In our previous work, we developed a mussel-inspired, catechol-functionalized hyaluronic acid (HA-CA) hydrogel that enabled effective cell transplantation due to its improved biocompatibility and strong tissue adhesiveness. The present study was performed to further expand the utility of HA-CA hydrogels for use in stem cell therapies to treat more clinically relevant tissue defect models. Specifically, we utilized HA-CA hydrogels to potentiate stem cell-mediated angiogenesis and osteogenesis in two tissue defect models: critical limb ischemia and critical-sized calvarial bone defect. HA-CA hydrogels were found to be less cytotoxic to human adipose-derived stem cells (hADSCs) in vitro compared to conventional photopolymerized HA hydrogels. HA-CA hydrogels also retained the angiogenic functionality of hADSCs and supported osteogenic differentiation of hADSCs. Because of their superior tissue adhesiveness, HA-CA hydrogels were able to mediate efficient engraftment of hADSCs into the defect regions. When compared to photopolymerized HA hydrogels, HA-CA hydrogels significantly enhanced hADSC-mediated therapeutic angiogenesis (promoted capillary/arteriole formation, improved vascular perfusion, attenuated ischemic muscle degeneration/fibrosis, and reduced limb amputation) and bone reconstruction (mineralized bone formation, enhanced osteogenic marker expression, and collagen deposition). This study proves the feasibility of using bioinspired HA-CA hydrogels as functional biomaterials for improved tissue regeneration in critical tissue defects.

Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres enhance large-wound healing by delivering growth factors in platelet-rich plasma.

Platelet-rich plasma (PRP) contains many growth factors that are involved in tissue regeneration processes. For successful tissue regeneration, protein growth factors require a delivery vehicle for long-term and sustained release to a defect site in order to maintain their bioactivity. Previously, we showed that heparin-conjugated poly(lactic-co-glycolic acid) nanospheres (HCPNs) can provide long-term delivery of growth factors with affinity for heparin. In this study, we hypothesize that treatment of a skin wound with a mixture of PRP and HCPNs would provide long-term delivery of several growth factors contained in PRP to promote the skin wound healing process with preservation of bioactivity. The release of platelet-derived growth factor-BB (PDGF-BB), contained in PRP, from HCPN with fibrin gel (FG) showed a prolonged release period versus a PRP mixture with FG alone (FG-PRP). Also, growth factors released from PRP with HCPN and FG showed sustained human dermal fibroblast growth for 12 days. Full-thickness skin wound treatment in mice with FG-HCPN-PRP resulted in much faster wound closure as well as dermal and epidermal regeneration at day 9 compared with treatment with FG-HCPN or FG-PRP. The enhanced wound healing using FG-HCPN-PRP may be due to the prolonged release not only of PDGF-BB but also of other growth factors in the PRP. The delivered growth factors accelerated angiogenesis at the wound site.

Identification of lactic acid bacteria in salted Chinese cabbage by SDS-PAGE and PCR-DGGE.

BACKGROUND: Lactic acid bacteria (LAB) in salted Chinese cabbage, the main ingredient of kimchi, were analyzed by culture-dependent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by sequencing of the 16S rRNA gene and by culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of the V3 region of the 16S rRNA gene. The results were compared to those of LAB that had previously been found in kimchi. RESULTS: The two identification methods produced distinct overall LAB profiles. The PCR-DGGE method detected a more diverse microflora, including non-LAB strains. The culture-dependent method uniquely detected Weissella sp. and was able to provide the quantitative distribution of LAB in samples. However, Leuconostoc mesenteroides, Lactobacillus curvatus and Leuconostoc carnosum, which had also been reported as the dominant LAB in kimchi in previous studies, were identified by both methods. CONCLUSION: The two identification methods gave different bacterial profiles, while both methods were sufficient to identify the most prevalent LAB in salted Chinese cabbage samples. The quantitative feature of the culture-dependent identification method would make it preferable for studying and monitoring LAB viability in kimchi at each fermentation stage. The availability of the culture-independent identification method to identify a broader bacterial profile, including non-LAB, would make it a more effective tool for controlling contamination of undesirable bacteria during kimchi fermentation.

Wet tissue adhesive polymeric powder hydrogels for skeletal muscle regeneration.

Volumetric muscle loss (VML) frequently results from traumatic incidents and can lead to severe functional disabilities. Hydrogels have been widely employed for VML tissue regeneration, which are unfortunately ineffective because of the lack of intimate contact with injured tissue for structural and mechanical support. Adhesive hydrogels allow for strong tissue connections for wound closure. Nevertheless, conventional adhesive hydrogels exhibit poor tissue adhesion in moist, bleeding wounds due to the hydration layer at the tissue-hydrogel interfaces, resulting in insufficient performance. In this study, we developed a novel, biocompatible, wet tissue adhesive powder hydrogel consisting of dextran-aldehyde (dex-ald) and gelatin for the regeneration of VML. This powder absorbs the interfacial tissue fluid and buffer solution on the tissue, spontaneously forms a hydrogel, and strongly adheres to the tissue via various molecular interactions, including the Schiff base reaction. In particular, the powder composition with a 1:4 ratio of dex-ald to gelatin exhibited optimal characteristics with an appropriate gelation time (258 s), strong tissue adhesion (14.5 kPa), and stability. Dex-ald/gelatin powder hydrogels presented strong adhesion to various organs and excellent hemostasis compared to other wet hydrogels and fibrin glue. A mouse VML injury model revealed that the dex-ald/gelatin powder hydrogel significantly improved muscle regeneration, reduced fibrosis, enhanced vascularization, and decreased inflammation. Consequently, our wet-adhesive powder hydrogel can serve as an effective platform for repairing various tissues, including the heart, muscle, and nerve tissues.

Rationally designed bioactive milk-derived protein scaffolds enhanced new bone formation.

Recently, a number of studies reported that casein was composed of various multifunctional bioactive peptides such as casein phosphopeptide and ß-casochemotide-1 that bind calcium ions and induce macrophage chemotaxis, which is crucial for bone homeostasis and bone fracture repair by cytokines secreted in the process. We hypothesized that the effects of the multifunctional biopeptides in casein would contribute to improving bone regeneration. Thus, we designed a tissue engineering platform that consisted of casein and polyvinyl alcohol, which was a physical-crosslinked scaffold (milk-derived protein; MDP), via simple freeze-thaw cycles and performed surface modification using 3,4-dihydroxy-l-phenylalanine (DOPA), a mussel adhesive protein, for immobilizing adhesive proteins and cytokines for recruiting cells in vivo (MDP-DOPA). Both the MDP and MDP-DOPA groups proved indirectly contribution of macrophages migration as RAW 264.7 cells were highly migrated toward materials by contained bioactive peptides. We implanted MDP and MDP-DOPA in a mouse calvarial defect orthotopic model and evaluated whether MDP-DOPA showed much faster mineral deposition and higher bone density than that of the no-treatment and MDP groups. The MDP-DOPA group showed the accumulation of host M2 macrophages and mesenchymal stem cells (MSCs) around the scaffold, whereas MDP presented mostly M1 macrophages in the early stage.

Fibrinogen-based cell and spheroid sheets manipulating and delivery for mouse hindlimb ischemia.

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higherin vitrotubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research.

Enhancement of long-term angiogenic efficacy of adipose stem cells by delivery of FGF2.

Stem cell transplantation can induce neovascularization. Regenerated blood vessels should remain stable for a long-term period in order to function as new blood vessels in ischemic tissues. Here we show that local delivery of FGF2 enhances the long-term (12weeks) angiogenic efficacy of human adipose-derived stem cells (hADSCs) implanted into mouse ischemic hindlimbs. Following transplantation of hADSCs into ischemic hindlimbs of mice, hADSC viability was significantly higher in the hADSC+FGF2 group at 4 and 12weeks post-transplantation than in the hADSC only group. Furthermore, hADSCs produced higher levels of angiogenic growth factors (i.e., fibroblast growth factor 2, vascular endothelial growth factor, hepatocyte growth factor, and platelet-derived growth factor) at both time points. As a result, the density of arterioles in the ischemic hindlimb muscle was significantly higher in the hADSC+FGF2 group than in either hADSC or FGF2 only group at both time points. The number of arterioles with larger diameters was significantly greater in the hADSC+FGF2 group than in the other groups at 12weeks, and increased in the hADSC+FGF2 group as the time period increased from 4weeks to 12weeks post-transplantation. This suggests that FGF2 delivery to hADSC transplantation sites enhances long-term angiogenic efficacy of hADSCs transplanted into ischemic tissues.

The effect of the delivery carrier on the quality of bone formed via bone morphogenetic protein-2.

Bone morphogenetic protein-2 (BMP-2) can induce bone generation in vivo. Although many studies have demonstrated an increased quantity of regenerated bone after the delivery of BMP-2 using various carriers, little is known about the effect of the carrier type on the quality of the regenerated bone. In this study, we compared the quality of regenerated bone when BMP-2 was delivered with either ß-tricalcium phosphate (ß-TCP) or heparin-conjugated fibrin (HCF), both of which are shown to be excellent carriers for BMP-2. The profile of the release of BMP-2 was not significantly different between the delivery carriers. However, the alkaline phosphate activity of cultured osteoblasts was significantly higher when BMP-2 was delivered using HCF than when BMP-2 was delivered using ß-TCP. To evaluate the quality of the regenerated bone, both types of BMP-2 carriers were implanted into critical-sized calvarial defects in mice. Eight weeks after implantation, the regenerated bone was examined by histomorphometry. Importantly, the treatment using HCF + BMP-2 and ß-TCP + BMP-2 resulted in similar bone formation areas. However, the treatment using HCF + BMP-2 resulted in significantly higher bone density than the treatment using ß-TCP + BMP-2. This study shows that a BMP-2 delivery carrier can modulate the quality of bone regenerated via BMP-2 delivery.

Skin regeneration with fibroblast growth factor 2 released from heparin-conjugated fibrin.

Fibroblast growth factor 2 (FGF2) stimulates skin wound healing but does long-term delivery of FGF2 enhance skin regeneration compared to short-term delivery? Heparin-conjugated fibrin (HCF) was used as a vehicle for long-term delivery of FGF2. Fibrin, HCF, FGF2-loaded fibrin, and FGF2-loaded HCF were implanted into full-thickness skin defects of mice. The neoepidermis thickness was significantly larger in the FGF2-loaded HCF group than in the other groups, except for the FGF2-loaded fibrin group. Suprabasal cytokeratin differentiation in squamous neoepithelium was greatest in the FGF2-loaded HCF group. The enhanced skin regeneration accompanying the long-term delivery of FGF2 could be mediated, at least partially, by enhanced neovascularization and cell proliferation in the neodermis.

Spinner-flask culture induces redifferentiation of de-differentiated chondrocytes.

Implantation of chondrocytes isolated from patients and expanded in number in vitro is being used to treat patients with cartilage injuries. However, chondrocytes de-differentiate during culture with several passages, and cartilage regenerated by implantation of de-differentiated chondrocytes may be suboptimal. Here, we show that a spinner-flask culture system induces formation of chondrocyte aggregates and redifferentiate de-differentiated chondrocytes. Spinner-flask cultures induced the aggregate formation of chondrocytes with passage 1 or 4. Importantly, spinner-flask cultures induced redifferentiation of the de-differentiated chondrocytes, as type I collagen expression was significantly lower and type II collagen expression was significantly higher in spinner flask-cultured chondrocytes than in monolayer-cultured chondrocytes. This system is easily scalable and could be feasible for clinical setting.

Chemoport with a non-collapsible chamber as a replacement for an Ommaya reservoir in the treatment of leptomeningeal carcinomatosis.

BACKGROUND: The Ommaya reservoir for intraventricular chemotherapy of leptomeningeal carcinomatosis (LMC) patients has been reported to have some complications. We introduced a Chemoport reservoir, with a solid non-collapsible, high-profile chamber as a the replacement for the Ommaya reservoir in LMC patients. OBJECTIVE: To evaluate the usefulness of Chemoport as an alternative to Ommaya for the intraventricular chemotherapy of LMC. METHODS: The medical records of 155 patients (89 Ommaya and 66 Chemoport) who underwent intraventricular chemotherapy via a subgaleal reservoir were reviewed. Chemoport was secured with engraving of skull. RESULT: Reservoir malfunction, including one intracranial hemorrhage (ICH) under the burr hole occurred, in six patients. During the course of therapy, cerebrospinal fluid (CSF) infection was diagnosed in 19 patients and intraventricular hemorrhage with ICH was evident in three patients of the Ommaya group. Incidence of the above-mentioned complications showed no difference between the two groups. CSF leakage under a galeal flap or through a wound edge occurred more frequently in the Ommaya group (12 patients) than in the Chemoport group (two patients) and the difference was statistically significant (p = 0.03). One-hundred and four patients showed increased intracranial pressure (ICP) and 74 of them received additional CSF drainage to control increased ICP by either intermittent CSF drainage in both groups or continuous extraventricular drainage (EVD) of CSF using designated hooked needle only in the Chemoport group. Among the factors related to the control ICP, the number of chemotherapies, type of reservoir in favor of Chemoport, and EVD showed significantly improved control of ICP (p < 0.05). CONCLUSION: Chemoport, as a reservoir for intraventricular chemotherapy, has superior ICP control at an equal or lower rate of complications compared with the Ommaya reservoir.

Improved spinal fusion efficacy by long-term delivery of bone morphogenetic protein-2 in a rabbit model.

BACKGROUND AND PURPOSE: Various new delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2) have been introduced to improve its efficacy in osteogenesis. Of these, we have previously developed heparin-conjugated PLGA nanospheres (HCPN) as a long-term delivery system for BMP-2. In vitro studies have shown that the BMP-2 long-term delivery system enhances the level of bone formation. However, the long-term effects of BMP-2 on spinal fusion have not been assessed. Therefore, we now tested the hypothesis that the long-term delivery of BMP-2 using HCPN improves spinal fusion compared to short-term delivery in a rabbit fusion model. METHODS: 24 adult New Zealand White rabbits underwent posterolateral fusion (6 animals in 4 groups). The autograft group received an autologous iliac chip bone graft as a positive control. The BMP-2-PN group received rhBMP-2 (20 µg per implant) and PLGA nanospheres (PN) suspended in fibrin gel, and served as a short-term release group. The HCPN group received HCPN suspended in fibrin gel without BMP-2 as a negative control. The BMP-2-HCPN group received rhBMP-2 (20 µg per implant)-bound HCPN suspended in fibrin gel and served as a long-term release group. All animals were killed 12 weeks after surgery. Manual palpation, axial tensile tests, radiography, and histological evaluations were then performed. RESULTS: The spinal fusion rate and Young's modulus of the fusion mass were better in the BMP-2 long-term delivery group than in the short-term delivery group at an equivalent dose. However, the outcome of the long-term delivery was inferior to that of the autograft group. INTERPRETATION: The HCPN system showed potential as an effective carrier that might improve the osteogenic efficacy of BMP-2 for spinal fusion.

Deep Learning Application in Spinal Implant Identification.

STUDY DESIGN: Retrospective observational study. OBJECTIVE: To demonstrate the clinical usefulness of deep learning by identifying previous spinal implants through application of deep learning. SUMMARY OF BACKGROUND DATA: Deep learning has recently been actively applied to medical images. However, despite many attempts to apply deep learning to medical images, the application has rarely been successful. We aimed to demonstrate the effectiveness and usefulness of deep learning in the medical field. The goal of this study was to demonstrate the clinical usefulness of deep learning by identifying previous spinal implants through application of deep learning. METHODS: For deep learning algorithm development, radiographs were retrospectively obtained from clinical cases in which the patients had lumbar spine one-segment instrument surgery. A total of 2894 lumbar spine anteroposterior (AP: 1446 cases) and lateral (1448 cases) radiographs were collected. Labeling work was conducted for five different implants. We conducted experiments using three deep learning algorithms. The traditional deep neural network model built by coding the transfer learning algorithm, Google AutoML, and Apple Create ML. Recall (sensitivity) and precision (specificity) were measured after training. RESULTS: Overall, each model performed well in identifying each pedicle screw implant. In conventional transfer learning, AP radiography showed 97.0% precision and 96.7% recall. Lateral radiography showed 98.7% precision and 98.2% recall. In Google AutoML, AP radiography showed 91.4% precision and 87.4% recall; lateral radiography showed 97.9% precision and 98.4% recall. In Apple Create ML, AP radiography showed 76.0% precision and 73.0% recall; lateral radiography showed 89.0% precision and 87.0% recall. In all deep learning algorithms, precision and recall were higher in lateral than in AP radiography. CONCLUSION: The deep learning application is effective for spinal implant identification. This demonstrates that clinicians can use ML-based deep learning applications to improve clinical practice and patient care.Level of Evidence: 3.

Enhanced extraction of skin interstitial fluid using a 3D printed device enabling tilted microneedle penetration.

Interstitial fluid (ISF) is a body fluid that fills, surrounds cells and contains various biomarkers, but it has been challenging to extract ISF in a reliable and sufficient amount with high speed. To address the issues, we developed the tilted microneedle ISF collecting system (TMICS) fabricated by 3D printing. In this system, the microneedle (MN) was inserted at 66° to the skin by TMICS so that the MN length could be extended within a safe range of skin penetration. Moreover, TMICS incorporating three MN patches created reliable ISF collecting conditions by penetrating the skin at consistent angle and force, 4.9 N. Due to the MN length increase and the patch number expansion, the surface area of the penetrated tissue was increased, thereby confirming that ISF extraction efficiency was improved. Skin ISF was collected into the paper reservoir on the patch, and the absorbed area was converted into a volume. ISF extraction from the rat skin in vivo by TMICS was well tolerated, and the 2.9 µL of ISF was obtained within 30 s. Therefore, TMICS is promising to apply in the diagnosis of multiple biomarkers in ISF with high speed and stability.

Antioxidant and anti-inflammatory activities of Prussian blue nanozyme promotes full-thickness skin wound healing.

Excessive reactive oxygen species (ROS) and unresolved inflammations are the major causes of impaired wound healing as they overwhelm the cellular antioxidant system and impede the healing process. In this study, we examined the application of Prussian blue (PB) nanozyme as a novel material for cutaneous wound healing through the alleviation of excessive ROS and inflammation modulation. The PB nanoparticles not only exhibited hydrogen peroxide (H2O2) degradation activity but also showed strong superoxide scavenging ability. PB nanozyme mitigated the intracellular ROS at a high oxidative stress environment, resulting in a pronounced cytoprotective effect. Moreover, PB nanozyme also displayed significant anti-inflammatory activity, as evident from the suppression of inflammatory mediators in the lipopolysaccharide (LPS) induced macrophage cells. Encouraged by the in vitro results, we evaluated the in vivo therapeutic efficacy of PB nanozyme in a full-thickness cutaneous wound model combined with LPS treatment to mimic bacterial infection. The beneficial effects of topically applied PB nanozyme on wound healing and tissue regeneration were evident compared to the control. The periodical administration of a low amount (50 µg × 4) of PB nanoparticles exhibited faster wound closure as well as collagen deposition, maturation, and organization. Moreover, the PB treatment effectively induced the differentiation of keratinocytes, enhanced the neovascularization, and reduced macrophage burden in the entire wound site. Thus, PB nanozyme not only accelerated the healing process in an infection-mimicking cutaneous wound model but also exhibited tissue regeneration characteristics owing to the synergistic effect of ROS-scavenging and anti-inflammatory activities.

Nanozyme Impregnated Mesenchymal Stem Cells for Hepatic Ischemia-Reperfusion Injury Alleviation.

Mesenchymal stem cell (MSC) based therapy holds great potential for treating numerous diseases owing to their capability to heal injured tissue/organs through paracrine factors secretion and immunomodulation. Despite the high hopes, the low viability of transplanted cells in the injured tissues due to the elevated oxidative stress levels remains the largest obstacle in MSC-based cell therapy. To achieve desired therapeutic efficiency, the survival of the transplanted MSCs in the high oxidative stress environment needs to be ensured. Herein, we proposed the use of a ROS-scavenging nanozyme to protect transplanted MSCs from oxidative stress-mediated apoptosis and thereby improve the therapeutic effect. Prussian blue (PB) nanoparticles as a biocompatible ROS-scavenging nanozyme were incorporated into the MSCs without affecting the stemness and differentiation potential of MSCs. The nanozyme impregnation significantly improved the survival of MSCs in a high oxidative stress condition as well as augmented their paracrine effect and anti-inflammatory properties, resulting in a profound therapeutic effect in vivo in the liver ischemia-reperfusion (I/R) injury animal model. Our results indicated that the nanozyme incorporation into MSCs is a simple but efficient way to improve the therapeutic potential of MSC-based cell therapy.

In situ pocket-type microcarrier (PMc) as a therapeutic composite: Regeneration of cartilage with stem cells, genes, and drugs.

We prepared pocket-type micro-carriers (PMc) with pores larger than 30 µm for use in cell delivery by adding 40 mg pluronic F-127 copolymers (F-127) to biodegradable PLGA dissolved in dichloromethane solution. The controlling the size of the pockets in this way facilitates the adhesion of cells by regulating the size of the pockets according to the cells having various sizes. The size of PMc pores could be controlled within a range of 2 to 30 µm by varying the F-127 content. The ratio of F-127 to DOPA-bPEI was most appropriate at 1: 1, and the pocket size at 10 mg/ml of F-127 was appropriate for adhering 20-30 µm stem cells. F-127 containing SOX9 pDNA, in combination with DOPA-polyethylene-coated gold nanoparticles and dexamethasone loaded in PMcs, promoted cartilage differentiation. Gold nanoparticles complex and dexamethasone (DEX) loaded in PMcs were identified by micro-CT imaging and fluorescence imaging, respectively. By captured in pore generated on/in microspheres, the stem cells were safe and stable for use in delivery, both in vitro and in an animal model. Thus, microsphere pores can safely capture stem cells, and at the same time provide a microenvironment in which the captured stem cells can differentiate into chondrocytes.

Locally delivered growth factor enhances the angiogenic efficacy of adipose-derived stromal cells transplanted to ischemic limbs.

Ischemia is a potentially fatal medical event that is associated with as many as 30% of all deaths. Stem cell therapy offers significant therapeutic promise, but poor survival following transplantation to ischemic tissue limits its efficacy. Here we demonstrate that nanosphere-mediated growth factor delivery can enhance the survival of transplanted human adipose-derived stromal cells (hADSCs) and secretion of human angiogenic growth factors per cell, and substantially improve therapeutic efficacy of hADSCs. In vitro, in hypoxic (1% oxygen) and serum-deprived conditions that simulate in vivo ischemia, fibroblast growth factor-2 (FGF2) significantly reduced hADSC apoptosis and enhanced angiogenic growth factor secretion. In vivo, hADSCs delivered intramuscularly into ischemic hind limbs in combination with FGF2 resulted in significant improvements in limb survival and blood perfusion, as well as survival of the transplanted hADSCs and secretion of human angiogenic growth factors (i.e., vascular endothelial growth factor, hepatocyte growth factor, and FGF2). Interestingly, the majority of transplanted hADSCs were localized adjacent to the microvessels rather than being incorporated into them, suggesting that their major contribution to angiogenesis might be to increase paracrine secretion of angiogenic growth factors. This study demonstrates the potential of hADSCs in combination with growth factors for use in the treatment of ischemia.

The efficacy of bone morphogenetic protein-2 depends on its mode of delivery.

Bone morphogenetic protein-2 (BMP-2) induces bone regeneration in a dose-dependent manner, with higher doses of BMP-2 inducing greater bone formation. Previously, we showed that long-term delivery of BMP-2 provides better ectopic bone formation than short-term delivery of an equivalent dose. In the present study, we investigated the efficacy of orthotopic bone formation over a range of BMP-2 doses, using different delivery modes. Heparin-conjugated poly(lactic-co-glycolic acid) nanospheres suspended in fibrin gel were used as a long-term delivery system, and fibrin gel was used as a short-term delivery system. Different doses of BMP-2 were delivered to mouse calvarial defects using either long-term or short-term delivery systems. Eight weeks after treatment, bone regeneration was evaluated by histomorphometry. For both delivery systems, bone regeneration increased as the BMP-2 dose increased up to 1 µg and did not increase beyond this dose. Importantly, at BMP-2 doses higher than 1 µg, long-term delivery resulted in much greater bone formation than short-term delivery. This study shows that long-term delivery of BMP-2 is more effective at enhancing orthotopic bone formation than short-term delivery over a range of doses.