Growth Factor Release Profiles Research Articles

Growth factor delivery through electrospun nanofibers in scaffolds for tissue engineering applications

Tissue engineering scaffolds should ideally mimic the natural ECM in structure and function. Electrospun nanofibrous scaffolds are easily fabricated and possess a biomimetic nanostructure. Scaffolds can mimic ECM function by acting as a depot for sustained release of growth factors. bFGF, an important growth factor involved in tissue repair and mesenchymal stem cell proliferation and differentiation, is a suitable candidate for sustained delivery from scaffolds. In this study, we present two types of PLGA nanofibers incorporated with bFGF, fabricated using the facile technique of blending and electrospinning (Group I) and by the more complex technique of coaxial electrospinning (Group II). bFGF was randomly dispersed in Group I and distributed as a central core within Group II nanofibers; both scaffolds showed similar protein encapsulation efficiency and release over 1-2 weeks. Although both scaffold groups favored bone marrow stem cell attachment and subsequent proliferation, cells cultured on Group I scaffolds demonstrated increased collagen production and upregulated gene expression of specific ECM proteins, indicating fibroblastic differentiation. The study shows that the electrospinning technique could be used to prolong growth factor release from scaffolds and an appropriately sustained growth factor release profile in combination with a nanofibrous substrate could positively influence stem cell behavior and fate.

Chondrogenic differentiation of mesenchymal stem cells embedded in a scaffold by long‐term release of TGF‐β3 complexed with chondroitin sulfate

In this study, mesenchymal stem cells (MSCs) embedded in biodegradable and water-swollen, elastic block copolymer scaffolds were assessed for MSC chondrogenesis. To determine the optimal conditions for chondrogenesis of the embedded rMSCs, transforming growth factor-beta 3 (TGF-beta 3) was physically conjugated with chondroitin sulfate (CS) and mixed into scaffolds, which were subsequently evaluated for the differentiation of transplanted rMSCs. In determination of CS-bound growth factors for chondrogenesis, scaffold mixed with rMSCs and TGF-beta 3 was then tested by growth factor release profiles, confocal laser microscopy, RT-PCR analysis, real time-QPCR, and histology. The results of several different analyses of the transplanted rMSCs embedded in the scaffolds showed that rMSCs coupled with a CS-bound TGF-beta 3 encapsulated scaffold evidenced superior cartilage tissue formation as measured by an assay of specific gene and protein expression. Moreover, the scaffold exhibited more rapid and more distinct morphology of differentiated rMSCs than was observed with other scaffolds, as determined by histology and immunochemical histology analysis. These results indicate that the elastic block copolymer scaffolds combined with a CS-bound TGF-beta 3 should prove very suitable matrix for cell-based cartilage tissue engineering.

Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants

The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with 125I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.

Heparin-Bound Transforming Growth Factor-β3 Enhances Neocartilage Formation by Rabbit Mesenchymal Stem Cells

Heparin binds growth factors to form a stable complex that maintains the biological activity and can retard the release pattern. To differentiate the embedded the stem cells, heparin-bind transforming growth factor (TGF)-beta3 was mixed with rabbit mesenchymal stem cells encapsulated with thermo-reversible hydrogel. It is suggested that the heparin-bound TGF-beta3 would help to increase the chondrogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel. To determine the optimal condition for neocartilage formation, we characterized hydrogel constructs with growth factor release profiles, confocal laser microscopy, reverse transcription-polymerase chain reaction analysis, and histology. Reverse transcription-polymerase chain reaction analysis of the resultant cartilage tissue revealed that a thermo-reversible hydrogels with a heparin-bound TGF-beta3 was optimal for cartilage tissue formation as measured by production of collagen Type II, aggrecan, and SOX9, and cartilage oligomeric matrix protein gene expression. Additionally, the proliferation rate and cartilage specific ECM production were both significantly greater in the presence of heparin-bound TGF-beta3 than in the control. The amount of cartilage-associated ECM proteins was examined by immunohistochemical staining (collagen type II), Safranin-O staining, and Alcian blue staining. These data indicate the potential use of heparin-binding TGFbeta-3 for reconstruction of neocartilage formation.

The Effects of Local bFGF Release and Uniaxial Strain on Cellular Adaptation and Gene Expression in a 3D Environment: Implications for Ligament Tissue Engineering

The objectives of this investigation were (1) to characterize the growth factor release profile of a basic fibroblast growth factor (bFGF)-coated three-dimensional (3D) polymer scaffold under static and cyclically strained conditions, and (2) to delineate the individual and collective contributions of locally released bFGF and mechanical strain on cellular morphology and gene expression in this 3D system. Scaffolds were treated with I(125)-bFGF and subjected to mechanical strain or maintained in a static environment and the media sampled for factor release over a period of 6 days. Over the first 10 hours, a burst release of 25% of the incorporated growth factor into the surrounding media was noted. At 24 hours, approximately 40% of the bFGF was released into the media, after which steady state was achieved and minimal subsequent release was noted. Mechanical stimulation had no effect on growth factor release from the scaffold in this system. To test the concerted effects of bFGF and mechanical stimulation on bone marrow stromal cells (BMSCs), scaffolds were loaded with 0, 100, or 500 ng of bFGF, seeded with cells, and subjected to mechanical strain or maintained in a static environment. Scaffolds were harvested at 1, 7, and 21 days for RT-PCR and histomorphometry. All scaffolds subjected to growth factor and/or mechanical stimulation demonstrated cellular adherence and spreading at 21 days. Conversely, in the absence of both bFGF and mechanical stimulation, cells demonstrated minimal cytoplasmic spread. Moreover, at 21 days, cells subjected to both mechanical stimulation and bFGF (500 ng) demonstrated the highest upregulation of stress-resistive (collagen I, III) and stress-responsive proteins (tenascin-C). The effect of growth factor may be dose sensitive, however, as unstrained scaffolds treated with 100 ng of bFGF demonstrated upregulation of gene expression comparable to strained scaffolds treated with lower doses of bFGF (0 or 100 ng). In conclusion, results from this study suggest that the stimulatory effects of bFGF are dose sensitive and appear to be influenced by the addition of mechanical strain. The concurrent application of biochemical and mechanical stimuli may be important in promoting the adaptation of BMSCs and driving the transcription of genes essential for synthesis of a functional ligament replacement tissue.

Comparison of Release Profiles of Various Growth Factors from Biodegradable Carriers

AbstractA biodegradable hydrogel was prepared by glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 as a carrier to release basic growth factors on the basis of polyion complexation. Basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein-2 (BMP-2) were sorbed from their aqueous solution into the dried gelatin hydrogels to prepare respective growth factor-incorporating hydrogels. Under an in vitro non-degradation condition, approximately 20 % of incorporated bFGF and TGF-β1 was released from the hydrogels within initial 40 min, followed by no further release, whereas a large initial release of BMP-2 was observed. After subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled growth factor in the mouse back, the remaining radioactivity was measured to estimate the in vivo release profile of growth factors. Incorporation into gelatin hydrogels enabled bFGF and TGF-β1 to retain in the body for about 15 days and the retention period well correlated with that of the gelatin hydrogel. Taken together, it is likely that the growth factors ionically complexed with acidic gelatin were released in vivo as a result of hydrogel biodegradation. On the contrary, basic BMP-2 did not ionically interact with acidic gelatin, resulting in no sustained released by the present biodegradable carrier system.