Strategies for Controlled Delivery of Growth Factors and Cells for Bone Regeneration

http://repositorium.sdum.uminho.pt/bitstream/1822/20642/1/file.pdf

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

Growth factors (GFs) are soluble proteins secreted by cells that have the ability to regulate a variety of cellular processes and tissue regeneration. However, their translation into clinical applications is limited due to their short effective half-life, low stability, and rapid inactivation by enzymes under physiological conditions. To maximize the effectiveness of GFs and their biologically relevant applicability, a wide variety of sophisticated bio-inspired systems have been developed that augment tissue repair and cellular regeneration by controlling how much, when, and where GFs are released. Recently, protein immobilization techniques combined with nanomaterial carriers have shown promise in mimicking the natural healing cascade during tissue regeneration by augmenting the delivery and effectiveness of GFs. This review evaluates the latest techniques in direct immobilization and relevant biomaterials used for GF loading and release, including synthetic polymers, albumin, polysaccharides, lipids, mesoporous silica-based nanoparticles (NPs), and polymeric capsules. Specifically, we focus on GF-encapsulated NPs in functionalized microporous scaffolds as a promising alternative with the ability to mimic extracellular matrix (ECM) hierarchical architectures and components with high cell affinity and bioactivity. Finally, we discuss how these next-generation, advanced delivery systems have been used to enhance tissue repair and regeneration and consider future implications for their use in the field of regenerative medicine. Nanomaterials can speed up the rate at which wounds heal and tissue regenerates. Songlin Peng from the Jinan University Second College of Medicine, China, and colleagues review the development of artificial materials that achieve this aim by mimicking the hierarchical architecture of the extracellular matrix. Cells can proliferate and migrate by secreting proteins that signal to adjacent cells. These proteins – known as growth factors – bind to specific receptors on the target cell. But it is difficult to harness this simple concept in clinical applications such as wound healing because the instability of growth factors limits their safety and cost effectiveness. Peng and co-workers review recent progress in the use of functionalized microporous scaffolds functionalized with growth factor encapsulated nanoparticles. They also outline its advantages over alternative approaches employing polymers, lipids and mesoporous silica-based nanoparticles. Schematic illustration of Biomaterial Strategies for Controlled Growth Factor (GF) Delivery for Biomedical Applications. (a) The direct approaches for the immobilization/encapsulation of GFs to biomaterials; (b) Nanocarriers for GFs encapsulation and release; (c) GFs encapsulated nanocarriers functionalized biomaterials for tissue regeneration.

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Novel biomaterial strategies for controlled growth factor delivery for biomedical applications

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) represent a promising cell-based therapy for a number of degenerative conditions. Many applications require cell expansion and involve the treatment of diseases and conditions found in an aging population. Therefore, the effects of donor age and long-term passage must be clarified. In this study, the effects of donor age and long-term passage on the morphology, proliferation potential, characteristics, mesodermal differentiation ability, and transdifferentiation potential of hMSCs towards neurogenic lineage were evaluated. Cells from child donors (0–12 years, n = 6) maintained their fibroblast-like morphology up to higher passages and proliferated in a greater number than those from adult (25–50 years, n = 6) and old (over 60 years, n = 6) donors. Adipogenic, osteogenic, and neurogenic differentiation potential decreased with age, while chondrogenic potential did not change. Long-term passage affected the morphology and proliferation of hMSCs from all ages. With increasing passage number, proliferation rate decreased and cells lost their typical morphology. Expression levels of neural markers (β III tubulin and NSE) and topo II isoforms in populations of nondifferentiated hMSCs were investigated by reverse transcription polymerase chain reaction analysis. While neural marker and topo IIβ expression levels increased due to increasing passage number in adult hMSCs compared to child hMSCs, topo IIα decreased in both. These results indicated that, even under highly standardized culture conditions, donor age and long-term passage have effects on hMSC characteristics, which should be taken into account prior to stem cell-based therapies.

Donor age and long-term culture affect differentiation and proliferation of human bone marrow mesenchymal stem cells.

The immune system plays key roles in tissue homeostatic and disease processes, and manipulation of innate and adaptive immune responses is of great promise for a wide array of human afflictions, including tissue repair and regeneration, cancer, autoimmune syndromes and chronic infections. Systemic approaches to immunomodulation can correct both hypoactive and hyperactive immunity; however, they typically interfere with the homeostatic role of the immune system at nontarget sites, are associated with lifelong comorbidities and potentially fatal side effects. To overcome these issues, macroscale delivery devices can be placed at sites of interest in the body and engineered to locally control the pharmacokinetics of immunomodulatory agents, including small molecules, macromolecules and cells. In this Review, we outline important cellular targets of immunotherapies in tissue repair and cancer and discuss how macroscale delivery devices can be designed to modulate the release of molecular factors to impact immune cell behaviour, control the fate of delivered therapeutic cells or directly recruit, house and modulate host cells for immunotherapy applications. Macroscale delivery devices can be used to manipulate innate and adaptive immune responses. In this Review, the authors highlight important cellular targets of immunotherapies in tissue repair and cancer and discuss macroscale biomaterials strategies for therapeutic immunomodulation.

Macroscale biomaterials strategies for local immunomodulation

Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, beta-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs.

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Age-dependent neuroectodermal differentiation capacity of human mesenchymal stromal cells: limitations for autologous cell replacement strategies

The present investigation was performed to study the bioactivity of osteoinductive and osteoproliferative growth factors after release from biocompatible bone implants. Three types of porous carriers were used in this study: hydroxyapatite, alpha tricalcium phosphate, and a neutralized glass ceramic. Implants were loaded with recombinant human bone morphogenetic protein 2 (rh-BMP-2) and recombinant human basic fibroblast growth factor (rh-bFGF) in a concentration of 2 μg/150 μL PBS each. The released growth factors were then applicated into SAOS-2-cell cultures. After 3, 5, and 7 days cell differentiation was measured by the activity of alkaline phosphatase (ALP), cell proliferation by using a MTT assay as well as a cell counter. Rh-BMP-2 released during the first hour from the scaffolds led to a significant increase of the activity of ALP in the incubated SAOS-2-cell culture after 3, 5, and 7 days. However, the incubation with rh-BMP-2 released after 24 h was not found to increase the expression of ALP. The incubation of cell cultures with rh-bFGF released during the first hour led to a significant increase of cell number and of extinction in the MTT assay, whereas this increase was not observed after incubation with rh-bFGF released after 24 h. The in vitro measured biological activity of released growth factors from the surface of synthetic implants is time-depending. If prolonged osteoinductive and osteoproliferative potency of growth factors is desired, a modified application technique should be chosen to stabilize those proteins. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008

Stefan Fickert

Papers

Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocols

Age-dependent neuroectodermal differentiation capacity of human mesenchymal stromal cells: limitations for autologous cell replacement strategies

Biological activity of recombinant human growth factors released from biocompatible bone implants.