Bone tissue, cellular, and molecular responses to titanium implants treated by anodic spark deposition

A myriad of titanium (Ti) surface modifications has been proposed to hasten the osseointegration. In this context, the aim of this study was to perform histomorphometric, cellular, and molecular analyses of the bone tissue grown in close contact with Ti implants treated by anodic spark deposition (ASD-AK). Acid-etched (AE) Ti implants either untreated or submitted to ASD-AK were placed into dog mandibles and retrieved at 3 and 8 weeks. It was noticed that both implants, AE and ASD-AK, were osseointegrated at 3 and 8 weeks. Histomorphometric analysis showed differences between treatments only for bone-to-implant contact, being higher on AE implants. Although not backed by histomorphometric results, gene expression of key bone markers was higher for bone grown in close contact with ASD-AK and for cells harvested from these fragments and cultured until subconfluence. Cell proliferation at days 7 and 10 and alkaline phosphatase activity at day 10 was higher on AE surfaces. No statistical significant difference was noticed for extracellular matrix mineralization at 17 days. Our results have shown that the Ti fixtures treated by ASD-AK allowed in vivo osseointegration and induced higher expression of key markers of osteoblast phenotype, suggesting that this surface treatment could be considered to produce implants for clinical applications. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:30923098, 2012.

Studio di biomateriali usati come scaffold per Tissue Engineering e loro caratterizzazione con tecniche spettroscopiche vibrazionali e di analisi termica

This research investigated someone of the main problems connected to the application of Tissue Engineering in the prosthetic field, in particular about the characterization of the scaffolding materials and biomimetic strategies adopted in order to promote the implant integration. The spectroscopic and thermal analysis techniques were usefully applied to characterize the chemico-physical properties of the materials such as – crystallinity; – relative composition in case of composite materials; – Structure and conformation of polymeric and peptidic chains; – mechanism and degradation rate; – Intramolecular and intermolecular interactions (hydrogen bonds, aliphatic interactions). This kind of information are of great importance in the comprehension of the interactions that scaffold undergoes when it is in contact with biological tissues; this information are fundamental to predict biodegradation mechanisms and to understand how chemico-physical properties change during the degradation process. In order to fully characterize biomaterials, this findings must be integrated by information relative to mechanical aspects and in vitro and in vivo behavior thanks to collaborations with biomedical engineers and biologists. This study was focussed on three different systems that correspond to three different strategies adopted in Tissue Engineering: biomimetic replica of fibrous 3-D structure of extracellular matrix (PCL-PLLA), incorporation of an apatitic phase similar to bone inorganic phase to promote biomineralization (PCL-HA), surface modification with synthetic oligopeptides that elicit the interaction with osteoblasts. The characterization of the PCL-PLLA composite underlined that the degradation started along PLLA fibres, which are more hydrophylic, and they serve as a guide for tissue regeneration. Moreover it was found that some cellular lines are more active in the colonization of the scaffold. In the PCL-HA composite, the weight ratio between the polymeric and the inorganic phase plays an essential role both in the degradation process and in the biomineralization of the material. The study of self-assembling peptides allowed to clarify the influence of primary structure on intermolecular and intermolecular interactions, that lead to the formation of the secondary structure and it was possible to find a new class of oligopeptides useful to functionalize materials surface. Among the analytical techniques used in this study, Raman vibrational spectroscopy played a major role, being non-destructive and non-invasive, two properties that make it suitable to degradation studies and to morphological characterization. Also micro-IR spectroscopy was useful in the comprehension of peptide structure on oxidized titanium: up to date this study was one of the first to employ this relatively new technique in the biomedical field.

Reconstructive Microsurgery and Tissue Engineering in Musculo-Skeletal Oncology - Innovative Techniques

Tumors involving bone and soft tissues are extremely challenging situations. With the recent advances of multi-modal treatment, not only the type of surgery has moved from amputation to limb-sparing procedures, but also the survivorship has improved considerably and reconstructive techniques have the goal to allow a considerably higher quality of life. In bone reconstruction, tissue engineering strategies are the main area of research. Re-vascularization and re-vitalisation of a massive allograft would considerably improve the outcome of biological reconstructions. Using a rabbit animal model, in this study we showed that, by implanting a vascular pedicle inside a weight bearing massive cortical allograft, the bone regeneration inside the allograft was higher compared to the non-vascularized implants, given the patency of the vascular pedicle. Improvement in the animal model and the addition of Stem Cells and Growth factors will allow a further improvement in the results. In soft tissue tumors, free and pedicled flaps have been proven to be of great help as reconstruction strategies. In this study we analyzed the functional and overall outcome of 14 patients who received a re-innervated vascularized flap. We have demonstrated that the use of the innovative technique of motor re-innervated muscular flaps is effective when the resection involves important functional compartments of the upper or lower limb, with no increase of post-operative complications. Although there was no direct comparison between this type of reconstruction and the standard non-innervated reconstruction, we underlined the remarkable high overall functional scores and patient satisfaction following this procedure.

Use of a 3D perfusion bioreactor with osteoblasts and osteoblast/endothelial cell co-cultures to improve tissue-engineered bone

The delivery of oxygen, nutrients, and the removal of waste are essential for cellular survival. Culture systems for 3D bone tissue engineering have addressed this issue by utilizing perfusion flow bioreactors that stimulate osteogenic activity through the delivery of oxygen and nutrients by low-shear fluid flow. It is also well established that bone responds to mechanical stimulation, but may desensitize under continuous loading. While perfusion flow and mechanical stimulation are used to increase cellular survival in vitro, 3D tissue-engineered constructs face additional limitations upon in vivo implantation. As it requires significant amounts of time for vascular infiltration by the host, implants are subject to an increased risk of necrosis. One solution is to introduce tissue-engineered bone that has been pre-vascularized through the co-culture of osteoblasts and endothelial cells on 3D constructs. It is unclear from previous studies: 1) how 3D bone tissue constructs will respond to partitioned mechanical stimulation, 2) how gene expression compares in 2D and in 3D, 3) how co-cultures will affect osteoblast activity, and 4) how perfusion flow will affect co-cultures of osteoblasts and endothelial cells. We have used an integrated approach to address these questions by utilizing mechanical stimulation, perfusion flow, and a co-culture technique to increase the success of 3D bone tissue engineering. We measured gene expression of several osteogenic and angiogenic genes in both 2D and 3D (static culture and mechanical stimulation), as well as in 3D cultures subjected to perfusion flow, mechanical stimulation and partitioned mechanical stimulation. Finally, we co-cultured osteoblasts and endothelial cells on 3D scaffolds and subjected them to long-term incubation in either static culture or under perfusion flow to determine changes in gene expression as well as histological measures of osteogenic and angiogenic activity. We discovered that 2D and 3D osteoblast cultures react differently to shear stress, and that partitioning mechanical stimulation does not affect gene expression in our model. Furthermore, our results suggest that perfusion flow may rescue 3D tissue-engineered constructs from hypoxic-like conditions by reducing hypoxia-specific gene expression and increasing histological indices of both osteogenic and angiogenic activity. Future research to elucidate the mechanisms behind these results may contribute to a more mature bone-like structure that integrates more quickly into host tissue, increasing the potential of bone tissue engineering.

An anisotropic elastic-viscoplastic damage model for bone tissue

A new anisotropic elastic-viscoplastic damage constitutive model for bone is proposed using an eccentric elliptical yield criterion and nonlinear isotropic hardening. A micromechanics-based multiscale homogenization scheme proposed by Reisinger et al. is used to obtain the effective elastic properties of lamellar bone. The dissipative process in bone is modeled as viscoplastic deformation coupled to damage. The model is based on an orthotropic ecuntric elliptical criterion in stress space. In order to simplify material identification, an eccentric elliptical isotropic yield surface was defined in strain space, which is transformed to a stress-based criterion by means of the damaged compliance tensor. Viscoplasticity is implemented by means of the continuous Perzyna formulation. Damage is modeled by a scalar function of the accumulated plastic strain D(κ) , reducing all element s of the stiffness matrix. A polynomial flow rule is proposed in order to capture the rate-dependent post-yield behavior of lamellar bone. A numerical algorithm to perform the back projection on the rate-dependent yield surface has been developed and implemented in the commercial finite element solver Abaqus/Standard as a user subroutine UMAT. A consistent tangent operator has been derived and implemented in order to ensure quadratic convergence. Correct implementation of the algorithm, convergence, and accuracy of the tangent operator was tested by means of strain- and stress-based single element tests. A finite element simulation of nano- indentation in lamellar bone was finally performed in order to show the abilities of the newly developed constitutive model.

A generalized anisotropic quadric yield criterion and its application to bone tissue at multiple length scales

Nonlinear computational analysis of materials showing elasto-plasticity or damage relies on knowledge of their yield behavior and strengths under complex stress states. In this work, a generalized anisotropic quadric yield criterion is proposed that is homogeneous of degree one and takes a convex quadric shape with a smooth transition from ellipsoidal to cylindrical or conical surfaces. If in the case of material identification, the shape of the yield function is not known a priori, a minimization using the quadric criterion will result in the optimal shape among the convex quadrics. The convexity limits of the criterion and the transition points between the different shapes are identified. Several special cases of the criterion for distinct material symmetries such as isotropy, cubic symmetry, fabric-based orthotropy and general orthotropy are presented and discussed. The generality of the formulation is demonstrated by showing its degeneration to several classical yield surfaces like the von Mises, Drucker–Prager, Tsai–Wu, Liu, generalized Hill and classical Hill criteria under appropriate conditions. Applicability of the formulation for micromechanical analyses was shown by transformation of a criterion for porous cohesive-frictional materials by Maghous et al. In order to demonstrate the advantages of the generalized formulation, bone is chosen as an example material, since it features yield envelopes with different shapes depending on the considered length scale. A fabric- and density-based quadric criterion for the description of homogenized material behavior of trabecular bone is identified from uniaxial, multiaxial and torsional experimental data. Also, a fabric- and density-based Tsai–Wu yield criterion for homogenized trabecular bone from in silico data is converted to an equivalent quadric criterion by introduction of a transformation of the interaction parameters. Finally, a quadric yield criterion for lamellar bone at the microscale is identified from a nanoindentation study reported in the literature, thus demonstrating the applicability of the generalized formulation to the description of the yield envelope of bone at multiple length scales.

The influence of yield surface shape and damage in the depth-dependent response of bone tissue to nanoindentation using spherical and Berkovich indenters

Prevention and treatment of osteoporosis rely on understanding of the micromechanical behaviour of bone and its influence on fracture toughness and cell-mediated adaptation processes. Postyield properties may be assessed by nonlinear finite element simulations of nanoindentation using elastoplastic and damage models. This computational study aims at determining the influence of yield surface shape and damage on the depth-dependent response of bone to nanoindentation using spherical and conical tips. Yield surface shape and damage were shown to have a major impact on the indentation curves. Their influence on indentation modulus, hardness, their ratio as well as the elastic-to-total work ratio is well described by multilinear regressions for both tip shapes. For conical tips, indentation depth was not statistically significant (p<0.0001). For spherical tips, damage was not a significant parameter (p<0.0001). The gained knowledge can be used for developing an inverse method for identification of postelastic properties of bone from nanoindentation.

Scaffolds, stem cells, and tissue engineering: A potent combination!

Stem cells, either from embryonic or adult sources, have demonstrated the potential to differentiate into a wide range of tissues depending on culture conditions. This makes them prime candidates for use in tissue engineering applications. Current technology allows us to process biocompatible and biodegradable polymers into three-dimensional (3D) configurations, either as solid porous scaffolds or hydrogels, with controlled macro and/or micro spatial geometry and surface chemistry. Such control provides us with the ability to present highly controlled microenvironments to a chosen cell type. However, the precise microenvironments required for optimal expansion and/or differentiation of stem cells are only now being elucidated, and hence the controlled use of stem cells in tissue engineering remains a very young field. We present here a brief review of the current literature detailing interactions between stem cells and 3D scaffolds of varying morphology and chemical properties, concluding with remaining challenges for those interested in tissue engineering using tailored scaffolds and stem cells.

Modelling oxygen diffusion and cell growth in a porous, vascularising scaffold for soft tissue engineering applications

Soft tissue engineering presents significant challenges compared to other tissue engineering disciplines such as bone, cartilage or skin engineering. The very high cell density in most soft tissues, often combined with large implant dimensions, means that the supply of oxygen is a critical factor in the success or failure of a soft tissue scaffold. A model is presented for oxygen diffusion in a 15-60 mm diameter dome-shaped scaffold fed by a blood vessel loop at its base. This model incorporates simple models for vascular growth, cell migration and the effect of cell density on the effective oxygen diffusivity. The model shows that the dynamic, homogeneous cell seeding method often employed in small-scale applications is not applicable in the case of larger scale scaffolds such as these. Instead, we propose the implantation of a small biopsy of tissue close to a blood supply within the scaffold as a technique more likely to be successful. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

An initial evaluation of gellan gum as a material for tissue engineering applications

Alpha-modified minimum essential medium (αMEM) has been found to cross-link a 1% gellan gum solution, resulting in the formation of a self-supporting hydrogel in 1:1 and 5:1 ratios of polysaccharide: αMEM. Rheological data from temperature sweeps confirm that in addition to orders of magnitude differences in G' between 1% gellan and 1% gellan with αMEM, there is also a 20°C increase in the temperature at which the onset of gelation takes place when αMEM is present. Frequency sweeps confirm the formation of a true gel; mechanical spectra for mixtures of gellan and αMEM clearly demonstrate G' to be independent of frequency. It is possible to immobilize cells within a three-dimensional (3D) gellan matrix that remain viable for up to 21 days in culture by adding a suspension of rat bone marrow cells (rBMC) in αMEM to 1% gellan solution. This extremely simple approach to cell immobilization within 3D constructs, made possible by the fact that gellan solutions cross-link in the presence of millimolar concentrations of cations, poses a very low risk to a cell population immobilized within a gellan matrix and thus indicates the potential of gellan for use as a tissue engineering scaffold. © 2007 Sage Publications.

Sea organisms as a source of collagen-derived polymers for skin tissue engineering applications

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin and they are usually accompanied by fibrotic reactions that result in the production of a scar. Natural biopolymers such as collagen have been a lot investigated as potential source of biomaterial for skin replacement in Tissue Engineering. Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role in connective tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches, as skin tissue engineering. In addition, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are skin and bone from bovine and porcine origin. However, these last carry high risk of bovine spongiform encephalopathy or transmissible spongiform encephalopathy and immunogenic responses. On the other hand, the increase of jellyfish has led us to consider this marine organism as potential collagen source for tissue engineering applications. In the present study, novel form of acid and pepsin soluble collagen were extracted from dried Rhopilema hispidum jellyfish species in an effort to obtain an alternative and safer collagen. We studied different methods of collagen purification (tissues and experimental procedures). The best collagen yield was obtained using pepsin extraction method (34.16 mg collagen/g of tissue). The isolated collagen was characterized by SDS-polyacrylamide gel electrophoresis and circular dichroism spectroscopy.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Translating tissue engineering technology platforms into cancer research

Technology platforms originally developed for tissue engineering applications produce valuable models that mimic three-dimensional (3D) tissue organization and function to enhance the understanding of cell/tissue function under normal and pathological situations. These models show that when replicating physiological and pathological conditions as closely as possible investigators are allowed to probe the basic mechanisms of morphogenesis, differentiation and cancer. Significant efforts investigating angiogenetic processes and factors in tumorigenesis are currently undertaken to establish ways of targeting angiogenesis in tumours. Anti-angiogenic agents have been accepted for clinical application as attractive targeted therapeutics for the treatment of cancer. Combining the areas of tumour angiogenesis, combination therapies and drug delivery systems is therefore closely related to the understanding of the basic principles that are applied in tissue engineering models. Studies with 3D model systems have repeatedly identified complex interacting roles of matrix stiffness and composition, integrins, growth factor receptors and signalling in development and cancer. These insights suggest that plasticity, regulation and suppression of these processes can provide strategies and therapeutic targets for future cancer therapies. The historical perspective of the fields of tissue engineering and controlled release of therapeutics, including inhibitors of angiogenesis in tumours is becoming clearly evident as a major future advance in merging these fields. New delivery systems are expected to greatly enhance the ability to deliver drugs locally and in therapeutic concentrations to relevant sites in living organisms. Investigating the phenomena of angiogenesis and anti-angiogenesis in 3D in vivo models such as the Arterio-Venous (AV) loop mode in a separated and isolated chamber within a living organism adds another significant horizon to this perspective and opens new modalities for translational research in this field.

Tissue engineering of articular cartilage with biomimetic zones

Articular cartilage damage is a persistent and increasing problem with the aging population, and treatments to achieve biological repair or restoration remain a challenge. Cartilage tissue engineering approaches have been investigated for over 20 years, but have yet to achieve the consistency and effectiveness for widespread clinical use. One of the potential reasons for this is that the engineered tissues do not have or establish the normal zonal organization of cells and extracellular matrix that appears critical for normal tissue function. A number of approaches are being taken currently to engineer tissue that more closely mimics the organization of native articular cartilage. This review focuses on the zonal organization of native articular cartilage, strategies being used to develop such organization, the reorganization that occurs after culture or implantation, and future prospects for the tissue engineering of articular cartilage with biomimetic zones.