Use Of Exogenous Growth Factors Research Articles

Toward the Development of a Tissue Engineered Gradient Utilizing CRISPR-Guided Gene Modulation.

Cellular, compositional, and mechanical gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have proven difficult due to the complex nature of these tissues. Current strategies for tissue engineering complex gradients often utilize stem cells; however, these multipotent cells require direction from environmental cues, which can be difficult to control both in vitro and in vivo. In this study, we utilize clustered regularly-interspaced short palindromic repeats (CRISPR)-guided gene modulation to direct the differentiation of multipotent adipose-derived stem cells (ASCs) to demonstrate the effectiveness of CRISPR-engineered cells in tissue engineering applications. Specifically, we screen CRISPR-interference (CRISPRi) constructs targeting the promotors of selected osteogenic inhibitors and demonstrate that ASC osteogenic differentiation and mineral deposition can be regulated with CRISPRi targeting of Noggin without the use of exogenous growth factors in tissue engineered constructs. As a proof of concept, we combine three technologies developed out of our laboratories to demonstrate the controlled deposition of these engineered cells in a gradient with CRISPR-activation multiplex-engineered aggrecan/collagen type-II-chondrogenic ASCs on a high density anisotropic type I collagen construct to create a cell and tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the enthesis. Our results display the promise of CRISPR-engineered ASCs to produce tissue gradients, similar to what is observed in native tissue.

Ovine Mesenchymal Stem Cell Chondrogenesis on a Novel 3D-Printed Hybrid Scaffold In Vitro.

This study evaluated the use of silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) 3D-printed scaffolds, with channel sizes of either 200 (SC-200) or 500 (SC-500) µm, as biomaterials to support the chondrogenesis of sheep bone marrow stem cells (oBMSC), under in vitro conditions. The objective was to validate the potential use of SiO2/PTHF/PCL-diCOOH for prospective in vivo ovine studies. The behaviour of oBMSC, with and without the use of exogenous growth factors, on SiO2/PTHF/PCL-diCOOH scaffolds was investigated by analysing cell attachment, viability, proliferation, morphology, expression of chondrogenic genes (RT-qPCR), deposition of aggrecan, collagen II, and collagen I (immunohistochemistry), and quantification of sulphated glycosaminoglycans (GAGs). The results showed that all the scaffolds supported cell attachment and proliferation with upregulation of chondrogenic markers and the deposition of a cartilage extracellular matrix (collagen II and aggrecan). Notably, SC-200 showed superior performance in terms of cartilage gene expression. These findings demonstrated that SiO2/PTHF/PCL-diCOOH with 200 µm pore size are optimal for promoting chondrogenic differentiation of oBMSC, even without the use of growth factors.

Degenerative tendon matrix induces tenogenic differentiation of mesenchymal stem cells

PurposeMesenchymal stem cells (MSCs) react dynamically with the surrounding microenvironment to promote tissue-specific differentiation and hence increase targeted regenerative capacity. Extracellular matrix (ECM) would be the first microenvironment to interact with MSCs injected into the tissue lesion. However, degenerative tissues would have different characteristics of ECM in comparison with healthy tissues. Therefore, the influence of degenerative ECM on tissue-specific differentiation of MSCs and the formation of matrix composition need to be considered for the sophisticated therapeutic application of stem cells for tissue regeneration.MethodsHuman degenerative tendon tissues were obtained from patients undergoing rotator cuff repair and finely minced into 2 ~ 3 mm fragments. Different amounts of tendon matrix (0.005 g, 0.01 g, 0.025 g, 0.05 g, 0.1 g, 0.25 g, 0.5 g, 1 g, and 2 g) were co-cultured with bone marrow MSCs (BM MSCs) for 7 days. Six tendon-related markers, scleraxis, tenomodulin, collagen type I and III, decorin, and tenascin-C, osteogenic marker, alkaline phosphatase (ALP), and chondrogenic marker, aggrecan (ACAN), were analyzed by qRT-PCR. Cell viability and senescence-associated beta-galactosidase assays were performed. The connective tissue growth factor was used as a positive control.ResultsThe expressions of six tendon-related markers were significantly upregulated until the amount of tendon matrix exceeded 0.5 g, the point where the mRNA expressions of all six genes analyzed started to decrease. The tendon matrix exerted an inhibitory effect on ACAN expression but had a negligible effect on ALP expression. Cell viability did not change significantly over the culture period. The amount of tendon matrix exceeding 0.01 g significantly increased the SA-βgal activity of BM MSCs.ConclusionThis study successfully demonstrated tendon ECM-stimulated tenogenesis of BM MSCs through an indirect co-culture system without the use of exogenous growth factors and the alteration of cellular viability. In contrast to the initial hypothesis, the tenogenesis of BM MSCs induced with the degenerative tendon matrix accompanied cellular senescence.

Alkaline activation of endogenous latent TGFβ1 by an injectable hydrogel directs cell homing for in situ complex tissue regeneration.

Utilization of the body's regenerative potential for tissue repair is known as in situ tissue regeneration. However, the use of exogenous growth factors requires delicate control of the dose and delivery strategies and may be accompanied by safety, efficacy and cost concerns. In this study, we developed, for the first time, a biomaterial-based strategy to activate endogenous transforming growth factor beta 1 (TGFβ1) under alkaline conditions for effective in situ tissue regeneration. We demonstrated that alkaline-activated TGFβ1 from blood serum, bone marrow fluids and soaking solutions of meniscus and tooth dentin was capable of increasing cell recruitment and early differentiation, implying its broad practicability. Furthermore, we engineered an injectable hydrogel (MS-Gel) consisting of gelatin microspheres for loading strong alkaline substances and a modified gelatin matrix for hydrogel click crosslinking. In vitro models showed that alkaline MS-Gel controllably and sustainably activated endogenous TGFβ1 from tooth dentin for robust bone marrow stem cell migration. More importantly, infusion of in vivo porcine prepared root canals with alkaline MS-Gel promoted significant pulp-dentin regeneration with neurovascular stroma and mineralized tissue by endogenous proliferative cells. Therefore, this work offers a new bench-to-beside translation strategy using biomaterial-activated endogenous biomolecules to achieve in situ tissue regeneration without the need for cell or protein delivery.

Nanocarrier-Mediated Topical Insulin Delivery for Wound Healing.

Wound care has been clinically demanding due to inefficacious treatment that represents an economic burden for healthcare systems. In Europe, approximately 7 million people are diagnosed with untreated wounds, leading to a cost between 6.000€ and 10.000€ per patient/year. In the United States of America, 1.5 million people over 65 years old suffer from chronic wounds. A promising therapeutic strategy is the use of exogenous growth factors because they are decreased at the wound site, limiting the recovery of the skin. Insulin is one of the cheapest growth factors in the market able to accelerate the re-epithelialization and stimulate angiogenesis and cell migration. However, the effectiveness of topical insulin in wound healing is hampered by the proteases in the wound bed. The encapsulation into nanoparticles improves its stability in the wound, providing adhesion to the mucosal surface and allowing its sustained release. The aim of this review is to perform a standing point about a promising strategy to treat different types of wounds by the topical delivery of insulin-loaded nanocarriers.

Response of the Injured Tendon to Growth Factors in the Presence or Absence of the Paratenon

Background: The paratenon is important for Achilles tendon healing. There is much interest in the use of exogenous growth factors (GFs) as potential agents for accelerating the healing of damaged Achilles tendons. Purpose/Hypothesis: The present study used a rat model to study the responses of the injured Achilles tendon to GFs in the presence or absence of the paratenon. The hypothesis was that responses of the injured tendon to GFs would be lower in the absence of a paratenon. Study Design: Controlled laboratory study. Methods: A 4-mm defect was created in the right Achilles tendon of 60 skeletally mature rats, which were treated with a validated combination of GFs (bFGF, BMP-12, and TGF-β1). Animals were randomly assigned to the intact paratenon (IP) group or resected paratenon (RP) group. Healing was studied anatomically, mechanically, and histologically after 1, 2, and 4 weeks. Results: IP tendons showed improved healing compared with RP tendons. IP tendons were significantly stronger (32.2 N and 48.9 N, respectively) than RP tendons (20.1 N and 31.1 N, respectively) after 1 and 2 weeks. IP tendons did not elongate as much as RP tendons and had greater cross-sectional areas (18.0 mm2, 14.4 mm2, and 16.4 mm2, respectively) after 1, 2, and 4 weeks compared with RP tendons (10.5 mm2, 8.4 mm2, and 11.9 mm2, respectively). On histology, earlier collagen deposition and parallel orientation of fibrils were found for IP tendons. Conclusion: The paratenon is essential for efficient Achilles tendon healing. Healing with GFs in this Achilles tendon defect model was superior in the presence of the paratenon. Clinical Relevance: Biological approaches to tendon engineering using GFs are in vogue and have been shown to improve healing of the rat Achilles tendon, most likely by inducing progenitor cells located within the paratenon. Clinically, resection or incision of the paratenon has been proposed for wound closure. Our data demonstrate the fundamental importance of the paratenon, which therefore should be preserved during Achilles tendon repair, especially if augmented with products such as platelet-rich plasma or autologous conditioned serum that are rich in GFs.

Apical Papilla Cells Are Capable of Forming a Pulplike Tissue with Odontoblastlike Cells without the Use of Exogenous Growth Factors

IntroductionDental pulp is a complex tissue with highly differentiated cells, which makes its reconstruction a challenging task. The apical papilla is an undifferentiated tissue considered as the remnant of the dental papilla that forms the dentin-pulp complex. Aiming to analyze morphologic features of the tissue formed in an in vivo pulp model, we used human apical papilla as a cell source without the use of exogenous growth factors. MethodsA construct was built using newborn mice molar crowns treated with TrypLE (Fisher Scientific, Loughborough, UK) and EDTA. The crowns were filled with PuraMatrix (Corning Inc, Corning, NY) and a pool population of human apical papilla cells. As a control, we used crowns filled only with PuraMatrix and empty crowns. The constructs were transplanted under severe combined immunodeficient mice kidney capsules. Immunohistochemistry for lamin A, dentin sialophosphoprotein, and dentin matrix protein 1 was performed. ResultsMorphologic analysis of all transplanted crowns showed the formation of a loose connective tissue of variable cellularity with the presence of well-formed functional vessels. In the study group, lamin A–positive cells represented the majority of cells within the pulp chamber and a few cells in the vessel lining. We also found positivity for dentin sialophosphoprotein and dentin matrix protein 1, an indicator of odontoblast differentiation. ConclusionsIn our study model, human transplanted apical papilla cells mixed with the host cells and formed a vascularized viable tissue, and these cells were able to differentiate into odontoblastlike cells without the use of exogenous growth factors.

Gold is for the mistress, silver for the maid: Enhanced mechanical properties, osteoinduction and antibacterial activity due to iron doping of tricalcium phosphate bone cements

Self-hardening calcium phosphate cements present ideal bone tissue substitutes from the standpoints of bioactivity and biocompatibility, yet they suffer from (a) weak mechanical properties, (b) negligible osteoinduction without the use of exogenous growth factors, and (c) a lack of intrinsic antibacterial activity. Here we attempt to improve on these deficiencies by studying the properties of self-setting Fe-doped bone-integrative cements containing two different concentrations of the dopant: 0.49 and 1.09 wt% Fe. The hardening process, which involved the transformation of Fe-doped β-tricalcium phosphate (Fe-TCP) to nanocrystalline brushite, was investigated in situ by continuously monitoring the cements using the Energy Dispersive X-Ray Diffraction technique. The setting time was 20 min and the hardening time 2 h, but it took 50 h for the cement to completely stabilize compositionally and mechanically. Still, compared to other similar systems, the phase transformation during hardening was relatively fast and it also followed a relatively simple reaction path, virtually free of complex intermediates and noisy background. Mössbauer spectrometry demonstrated that 57Fe atoms in Fe-TCP were located in two non-equivalent crystallographic sites and distributed over positions with a strong crystal distortion. The pronounced presence of ultrafine crystals in the final, brushite phase contributed to the reduction of the porosity and thereby to the enhancement of the mechanical properties. The compressive strength of the hardened TCP cements increased by more than twofold when Fe was added as a dopant, i.e., from 11.5 ± 0.5 to 24.5 ± 2.0 MPa. The amount of iron released from the cements in physiological media steadied after 10 days and was by an order of magnitude lower than the clinical threshold that triggers the toxic response. The cements exhibited osteoinductive activity, as observed from the elevated levels of expression of genes encoding for osteocalcin and Runx2 in both undifferentiated and differentiated MC3T3-E1 cells challenged with the cements. The osteoinductive effect was inversely proportional to the content of Fe ions in the cements, indicating that an excessive amount of iron can have a detrimental effect on the induction of bone growth by osteoblasts in contact with the cement. In contrast, the antibacterial activity of the cement in the agar assay increased against all four bacterial species analysed (E. coli, S. enteritidis, P. aeruginosa, S. aureus) in direct proportion with the concentration of Fe ions in it, indicating their key effect on the promotion of the antibacterial effect in this material. This effect was less pronounced in broth assays. Experiments involving co-incubation of cements with cells in an alternate magnetic radiofrequency field for 30 min demonstrated a good potential for the use of these magnetic cements in hyperthermia cancer therapies. Specifically, the population of human glioblastoma cells decreased six-fold at the 24 h time point following the end of the magnetic field treatment, while the population of the bone cancer cells dropped approximately twofold. The analysis of the MC3T3-E1 cell/cement interaction reiterated the effects of iron in the cement on the bone growth marker expression by showing signs of adverse effects on the cell morphology and proliferation only for the cement containing the higher concentration of Fe ions (1.09 wt%). Biological testing concluded that the effects of iron are beneficial from the perspective of a magnetic hyperthermia therapy and antibacterial prophylaxis, but its concentration in the material must be carefully optimized to avoid the adverse effects induced above a certain level of iron concentrations.

Functional tissue-engineered microtissue derived from cartilage extracellular matrix for articular cartilage regeneration

We developed a promising cell carrier prepared from articular cartilage slices, designated cartilage extracellular matrix (ECM)-derived particles (CEDPs), through processes involving physical pulverization, size screening, and chemical decellularization. Rabbit articular chondrocytes (ACs) or adipose-derived stem cells (ASCs) rapidly attached to the surface of the CEDPs and proliferated with high cell viability under microgravity (MG) condition in a rotary cell culture system (RCCS) or static condition. Gene profiling results demonstrated that ACs expanded on CEDPs exhibited significantly enhanced chondrogenic phenotypes compared with monolayer culture, and that ASCs differentiated into a chondrogenic phenotype without the use of exogenous growth factors. Moreover, MG culture conditions in a RCCS bioreactor were superior to static culture conditions in terms of maintaining the chondrogenic phenotype of ACs and inducing ACS chondrogenesis. With prolonged expansion, functional microtissue aggregates of AC- or ASC-laden CEDPs were formed. Further, AC- or ASC-based microtissues were directly implanted in vivo to repair articular osteochondral defects in a rabbit model. Histological results, biomechanical evaluations, and radiographic assessments indicated that AC- and ASC-based microtissues displayed equal levels of superior hyaline cartilage repair, whereas the other two treatment groups, in which osteochondral defects were treated with CEDPs alone or fibrin glue, exhibited primarily fibrous tissue repair. These findings provide an alternative method for cell culture and stem cell differentiation and a promising strategy for constructing tissue-engineered cartilage microtissues for cartilage regeneration. Statement of SignificanceDespite the remarkable progress in cartilage tissue engineering, cartilage repair still remains elusive. In the present study, we developed a cell carrier, namely cartilage extracellular matrix-derived particles (CEDPs), for cell proliferation of articular chondrocytes (ACs) and adipose-derived stem cells (ASCs), which improved the maintenance of chondrogenic phenotype of ACs, and induced chondrogenesis of ASCs. Moreover, the functional microtissue aggregates of AC- or ASC-laden CEDPs induced equal levels of superior hyaline cartilage repair in a rabbit model. Therefore, our study demonstrated an alternative method for chondrocyte culture and stem cell differentiation, and a promising strategy for constructing tissue-engineered cartilage microtissues for in vivo articular cartilage repair and regeneration.

Co-culture of adipose-derived stem cells and chondrocytes on three-dimensionally printed bioscaffolds for craniofacial cartilage engineering.

Reconstruction of craniofacial cartilagenous defects are among the most challenging surgical procedures in facial plastic surgery. Bioengineered craniofacial cartilage holds immense potential to surpass current reconstructive options, but limitations to clinical translation exist. We endeavored to determine the viability of utilizing adipose-derived stem cell-chondrocyte co-culture and three-dimensional (3D) printing to produce 3D bioscaffolds for cartilage tissue engineering. We describe a feasibility study revealing a novel approach for cartilage tissue engineering with in vitro and in vivo animal data. Porcine adipose-derived stem cells and chondrocytes were isolated and co-seeded at 1:1, 2:1, 5:1, 10:1, and 0:1 experimental ratios in a hyaluronic acid/collagen hydrogel in the pores of 3D-printed polycaprolactone scaffolds to form 3D bioscaffolds for cartilage tissue engineering. Bioscaffolds were cultured in vitro without growth factors for 4 weeks and then implanted into the subcutaneous tissue of athymic rats for an additional 4 weeks before sacrifice. Bioscaffolds were subjected to histologic, immunohistochemical, and biochemical analysis. Successful production of cartilage was achieved using a co-culture model of adipose-derived stem cells and chondrocytes without the use of exogenous growth factors. Histology demonstrated cartilage growth for all experimental ratios at the post-in vivo time point confirmed with type II collagen immunohistochemistry. There was no difference in sulfated-glycosaminoglycan production between experimental groups. Tissue-engineered cartilage was successfully produced on 3D-printed bioresorbable scaffolds using an adipose-derived stem cell and chondrocyte co-culture technique. This potentiates co-culture as a solution for several key barriers to a clinically translatable cartilage tissue engineering process. NA. Laryngoscope, 128:E251-E257, 2018.

Chondroinductive factor-free chondrogenic differentiation of human mesenchymal stem cells in graphene oxide-incorporated hydrogels.

Graphene-based nanomaterials have been applied as biomaterials to enhance stem cell adhesion, growth and differentiation by serving as nanocarriers for growth factors or other small molecules. However, the direct effect of graphene oxide (GO) itself on stem cells, in the absence of exogenous differentiation inductive factors, has not been tested. In this study, we loaded GO nanosheets and human bone marrow-derived mesenchymal stem cells (hBMSC) into a photopolymerizable poly-d,l-lactic acid/polyethylene glycol (PDLLA) hydrogel, a robust chondrosupportive scaffold recently developed in our laboratory, and assessed hBMSC differentiation along the chondrogenic lineage without supplemental chondroinductive factors. We first examined the effect of GO incorporation on the mechanical properties of constructs, and observed that the GO-containing constructs (GO/PDLLA) exhibited enhanced compressive modulus in a GO concentration dependent manner. hBMSCs cultured in GO/PDLLA maintained high cell viability (>95%), indicating minimal cytotoxicity of GO. Importantly, compared to those encapsulated in PDLLA hydrogel, hBMSCs within GO/PDLLA showed significantly higher level of gene expression of the cartilage matrix genes, aggrecan and collagen type II, and produced more cartilage matrix. In addition, the pro-chondrogenesis effect of GO increased with increasing GO concentration. Immunohistochemical results suggested that GO-enhanced hBMSC chondrogenesis was correlated with enriched sequestration of insulin, a necessary supplement known to have pro-chondrogenesis effects on hBMSC. Taken together, these findings demonstrate the utility of using GO to improve the mechanical properties and chondrogenic differentiation state of MSC-laden, engineered hydrogel constructs, without the use of exogenous growth factors, thus representing a potentially promising, biologics-free approach for cartilage tissue engineering.

Induction of mesenchymal stem cell chondrogenic differentiation and functional cartilage microtissue formation for in vivo cartilage regeneration by cartilage extracellular matrix-derived particles

We propose a method of preparing a novel cell carrier derived from natural cartilage extracellular matrix (ECM), designated cartilage ECM-derived particles (CEDPs). Through a series of processes involving pulverization, sieving, and decellularization, fresh cartilage was made into CEDPs with a median diameter of 263±48μm. Under microgravity culture conditions in a rotary cell culture system (RCCS), bone marrow stromal cells (BMSCs) can proliferate rapidly on the surface of CEDPs with high viability. Histological evaluation and gene expression analysis indicated that BMSCs were differentiated into mature chondrocytes after 21days of culture without the use of exogenous growth factors. Functional cartilage microtissue aggregates of BMSC-laden CEDPs formed as time in culture increased. Further, the microtissue aggregates were directly implanted into trochlear cartilage defects in a rat model (CEDP+MSC group). Gait analysis and histological results indicated that the CEDP+MSC group obtained better and more rapid joint function recovery and superior cartilage repair compared to the control groups, in which defects were treated with CEDPs alone or only fibrin glue, at both 6 and 12weeks after surgery. In conclusion, the innovative cell carrier derived from cartilage ECM could promote chondrogenic differentiation of BMSCs, and the direct use of functional cartilage microtissue facilitated cartilage regeneration. This strategy for cell culture, stem cell differentiation and one-step surgery using cartilage microtissue for cartilage repair provides novel prospects for cartilage tissue engineering and may have further broad clinical applications. Statement of SignificanceWe proposed a method to prepare a novel cell carrier derived from natural cartilage ECM, termed cartilage ECM-derived particles (CEDPs), which can support proliferation of MSCs and facilitate their chondrogenic differentiation. Further, the direct use of functional cartilage microtissue of MSC-laden CEDP aggregates for cartilage repair in vivo induced hyaline-like articular cartilage repair. This strategy for cell culture, stem cell differentiation and the one-step surgery for cartilage repair provide novel prospects for cartilage tissue engineering and may have further broad clinical applications.

Integrin specificity as a novel strategy for enhancing transplanted stem cell survival and tissue repair in vivo

Event Abstract Back to Event Integrin specificity as a novel strategy for enhancing transplanted stem cell survival and tissue repair in vivo Amy Clark1, 2* and Andres J. Garcia1, 2 1 Georgia Institute of Technology, Woodruff School of Mechanical Engineering, United States 2 Georgia Institute of Technology, Petit Institute for Bioengineering and Bioscience, United States Introduction: Despite the promising clinical results for the use of human mesenchymal stem cells (hMSC) in musculoskeletal defect treatment, inadequate control over cell survival, engraftment and fate limits the success of this cell-based therapy. Integrin-mediated cell adhesion plays a central role in tissue formation, maintenance, and repair by providing anchorage forces and triggering signals that regulate cell function. We hypothesize biomaterials presenting integrin-specific adhesive motifs will direct hMSC signaling and specification. The objective of this project is to engineer bioartificial hydrogels presenting integrin-specific ligands as biomimetic cell delivery vehicles for enhanced in vivo engraftment and function – an innovative strategy as it focuses on engineering specificity to integrin receptors to promote survival and cell-based repair without the use of exogenous growth factors. Methods: Early passage hMSC (<4) were transduced to constitutively express firefly luciferase (hMSCFLuc). 4-arm PEG-maleimide was functionalized with one of three ligands: α2β1-binding collagen I-mimetic GFOGER, αvβ3-binding RGD, or non-adhesive RDG peptides and cross-linked with a protease-degradable peptide. 2.5 mm defects were created in the radii of 8-9 week old male NOD scid gamma mice and treated with cell-laden hydrogels containing 15k hMSC. Bioluminescence was monitored using an In Vivo Imaging System (PerkinElmer). New bone formation was evaluated using an in vivo microcomputer tomography scanner (Scanco Medical). Gene expression was evaluated after 1 week in vivo. Total RNA was extracted from cells in the defect space and standardized across all samples. 96 total genes were screened using Fluidigm gene chip technology. Results: Bioluminescence of transplanted hMSCFLuc was monitored after transplantation (Fig 1A). The significantly higher bioluminescent signal in GFOGER/hMSCFLuc-treated defects suggests GFOGER hydrogels support transplanted cell survival and proliferation in vivo whereas the bioluminescent signals for hMSCFLuc delivered in a RGD or RDG hydrogel remained low. GFOGER/hMSC-treated defects exhibited significantly higher bone volume at weeks 4 and 8 compared to RGD and RDG with no statistical difference between groups receiving hMSCFLuc and hMSC (Fig 1B). Differences in gene expression for approximately 60 genes were observed after 1 week in vivo, with statistically higher expression of vascularization and inflammation genes in GFOGER/hMSC treated defects (Fig 2). Conclusions: GFOGER-functionalized PEG hydrogels support enhanced bone formation and hMSC viability in vivo compared to RGD and RDG, possibly via enhanced vascularization These studies are ongoing for immunohistochemical, cytokine, and defect vascularization analyses. This work highlights integrin-specificity as an important consideration in the design of cell delivery vehicles for engraftment and tissue repair. NIH grants R01 AR062368 and R01 AR062920

Molecular mechanism of hypoxia-induced chondrogenesis and its application in in vivo cartilage tissue engineering

Cartilage engineering is one of the most challenging issue in regenerative medicine, due to its limited self-ability to repair. Here, we assessed engineering of cartilage tissue starting from human bone marrow (hBM) stem cells under hypoxic environment and delineated the mechanism whereby chondrogenesis could be conducted without addition of exogenous growth factors. hBM stem cells were cultured in alginate beads and chondrogenesis was monitored by chondrocyte phenotypic markers. Activities and roles of Sox and HIF-1α transcription factors were investigated with complementary approaches of gain and loss of function and provided evidences that HIF-1α is essential for hypoxic induction of chondrogenesis. Thereafter, hBM cells and human articular chondrocytes (HAC) underwent chondrogenesis by 3D and hypoxic culture for 7 days or by ectopic expression of HIF-1α. After subcutaneous implantation of 3 weeks into athymic mice, tissue analysis showed that hypoxia or HIF-1α overexpression is effective and sufficient to induce chondrocyte phenotype in hBM cells, without use of exogenous growth factors. Therefore, this study brings interesting data for a simple and affordable system in biotechnology of cartilage engineering.

Poly(methacrylic acid‐co‐methyl methacrylate) beads promote vascularization and wound repair in diabetic mice

Topical application of beads made from poly(methacrylic acid-co-methyl methacrylate) (45 mol % methacrylic acid, MAA) increased the number of blood vessels and improved 1.5 x 1.5 cm full thickness wound closure in a diabetic mouse (db/db) model. Three groups were compared: MAA beads, control poly(methyl methacrylate) beads (PMMA), and no bead blanks. MAA bead treatment significantly increased percent wound closure at all timepoints (7, 14, and 21 days) with MAA bead-treated wounds almost closed at day 21 (91 +/- 5.4% MAA vs. 79 +/- 3.2% PMMA or 76 +/- 4.8% no beads; p < 0.05). This was consistent with the expected significant increase in vascularity in the MAA group at days 7 and 14. For example at day 14, MAA bead-treated wounds had a vascular density of 22.7 +/- 2.6 vessels/hpf compared with 17.0 +/- 2.0 vessels/hpf in the PMMA bead group (p < 0.05). Epithelial gap and migration measurements suggested that the increased vascularity leads to enhanced epithelial cell migration as a principal means of wound closure. Although studies are underway to elucidate the mechanism of this angiogenic response, the results presented here support the notion that such materials, perhaps in other forms, may be useful in wound care or in other situations where vascularity is to be enhanced without the use of exogenous growth factors.

Temporal expression of growth factors and matrix molecules in healing tendon lesions

Overuse tendon injuries are common among elite and recreational athletes. Tendon healing may be enhanced at the cellular level through the use of exogenous growth factors; however, little is known about the endogenous expression of growth factors in healing tendon. This study describes the temporal expression of insulin-like growth factor-I (IGF-I), transforming growth factor-β1 (TGF-β1), and collagen types I and III in healing tendon lesions. Collagenase-induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of 14 horses. Tendons were harvested from euthanatized horses 1, 2, 4, 8 or 24 weeks following injury. Gene expression was evaluated using Northern blot analysis (collagen types I and III), real time PCR (IGF-I and TGF-β1), and in situ hybridization. Protein content was assayed by dye-binding assay (collagen types I and III), radioimmunoassay (IGF-I), ELISA (TGF-β1), and immunohistochemistry. Samples were also processed for differential collagen typing, DNA and glycosaminoglycan content, and routine H&E staining. Microscopically, lesions progressed from an amorphous, acellular lesion soon after injury to scar tissue filled with collagen fibers and mature fibroblasts organized along lines of tension. Early lesions were characterized by immediate increases in expression of growth factors and collagen. Message levels for TGF-β1 peaked early in the wound healing process (1 week), while IGF-I peaked later (4 weeks), as the regenerative phase of healing was progressing. In the first 2 weeks after lesion induction, tissue levels of IGF-I protein actually decreased approximately 40% compared to normal tendon. By 4 weeks, these levels had exceeded those of normal tendon and remained elevated through 8 weeks. Message expression for collagen types I and III increased by 1 week following injury and remained elevated throughout the course of the study. Collagen type I represented the major type of collagen in healing tendon at all time points of the study. Based on these results, IGF-I, administered exogenously during the first 2 weeks following injury, may provide a therapeutic advantage by bolstering low endogenous tissue levels and enhancing the metabolic response of individual tendon fibroblasts.

Effects of basic fibroblast growth factor (FGF-2) on proliferation of human skin fibroblasts in type II diabetes mellitus.

Skin fibroblasts from patients with diabetes mellitus display abnormalities in cell proliferation. The use of exogenous growth factors on diabetic wounds has been found to stimulate fibroblast proliferation and facilitate wound healing. However, the results of application of FGF-2 alone to diabetic wounds in clinical trials have been disappointing. The objective of this experiment was to study the effects of FGF-2 and media supplements on in vitro proliferation of skin fibroblasts from patients with type II diabetes and nondiabetic controls, and to evaluate the association between fibroblast proliferation and cAMP production. Fibroblast cell lines (n = 5 from diabetic and n = 5 from control individuals) were cultured in DMEM + 20% FBS for 7 days. Cells were then counted, plated into 24-well plates at a concentration of 2 x 10(4) cells/well and incubated for 24 h in DMEM with serum. The next day, medium was changed to serum-free DMEM alone or DMEM with supplements (albumin, transferrin, insulin and hydrocortisone). Cells were cultured in the presence or absence of varying doses of FGF-2 (0, 0.3, 1, 3, 10 and 30 ng/ml) for 72 hrs then counted and medium was collected for cAMP radioimmunoassay. The doubling time for cell number tended to be greater (p < 0.2) for diabetic fibroblasts than for control fibroblasts. The addition of supplements to the medium reduced (p < 0.05) the doubling time for both fibroblast types. FGF-2 stimulated (p < 0.05) proliferation of diabetic fibroblasts only in medium containing supplements. In contrast, FGF-2 stimulated proliferation of control fibroblasts in medium with or without supplements. The maximal effects of FGF-2 on fibroblast proliferation were greater (p < 0.02) in medium with supplements than in medium without supplements. The K(D) of FGF-2 for fibroblast proliferation was greater (p < 0.06) for diabetic than for control fibroblasts, and lower (p < 0.02) for medium with supplements than for medium without supplements. Fibroblasts from patients with diabetes mellitus produced more (p < 0.05) cAMP than control fibroblasts. These results demonstrate that FGF-2 requires the presence of supplements to enhance proliferation of fibroblasts from patients with type II diabetes mellitus. In addition, fibroblasts from diabetic patients showed a greater K(D) for FGF-2 in terms of cell proliferation. These data suggest a defective FGF receptor or down-regulation of the FGF receptor-mediated cascade that leads to cell proliferation. Identifying methods of reducing the K(D) of FGF-2 in stimulating the proliferation of diabetic fibroblasts may improve the clinical response of diabetic wounds to FGF-2.

New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments.

A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin. Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin. To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed. In the short-term culture experiments, it was found that cubes of PVF (10 x 10 x 2 mm and 130 microm in pore size) coated with type I collagen with a seeding density of 2x10(7) BM cells offered the most appropriate culture conditions. In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks. In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer. In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture. Based upon the results of in vitro experiments, in vivo transplantation studies were also performed. Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix. Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients. From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation.

Opportunistic fusarial infections in humans.

Fusarium species are common hyaline soil saprophytes and plant pathogens which have frequently been reported as etiologic agents of opportunistic infections in humans. These infections have usually been limited to superficial mycoses, but recently the number of infections of deep tissues and disseminated infections has greatly increased, especially in patients with an underlying immunosuppressive condition. The characteristic signs of these infections are disseminated skin nodules, fungemia and multiorgan involvement. Frequently, myalgia is also present. Skin involvement occurred in over 80% of cases of disseminated infections. These lesions are significant because they are readily accessible for biopsy and culture, thus permitting an early diagnosis. The therapy and outcome are dependent on the degree of invasion of the organisms and the status of the host. Identification of the pathogen to genus level is not difficult, but identification to species level requires a greater degree of expertise. Up to now, 15 species of Fusarium have been reported to cause infections in humans and animals. Few patients with disseminated fusarial infections have survived, even after receiving an adequate dosage of amphotericin B, the only antifungal agent that has some effect against these fungi. In vitro susceptibility to amphotericin B is a poor predictor of the clinical outcome of invasive fungal infections. Recovery of the phagocytic mechanisms in the form of rising neutrophil counts appears to be mandatory for clinical resolution. The resolution of neutropenia may be aided by the use of exogenous growth factors. Outside the USA, the majority of cases of disseminated fusarial infection have been reported from Mediterranean or tropical countries.

Wound healing. The role of platelet-derived growth factor and transforming growth factor beta.

Recent investigation into the mechanisms of wound healing has indicated the interaction of many substances, including several growth factors. The activity of platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), are best defined. Both factors are secreted primarily from the alpha granules of platelets, but also from activated macrophages and fibroblasts. Investigation implicates the platelet as the initiator of wound healing, secreting PDGF, TGF-beta, and other factors that are chemotactic for monocytes, macrophages, and fibroblasts. Although their mode of action and degree of effect are different, both PDGF and TGF-beta increase the collagen content and early rate of gain of strength in wounds in normal and compromised tissue. In normal tissue, however, there is no long-term effect on wound outcome. The use of exogenous growth factors offers potential for chemical manipulation of the healing wound, particularly in tissues that are compromised, or where healing is abnormal.