Native Extracellular Matrix Components Research Articles

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypicalternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Rat liver extracellular matrix and perfusion bioreactor culture promote human amnion epithelial cell differentiation towards hepatocyte-like cells

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells’ maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

Determining the optimal pancreatic decellularization protocol, taking into account tissue morphological features

Introduction. Developing a tissue-engineered pancreatic construct (TEPC) involves a search for matrices/scaffolds capable of mimicking the structure and composition of the natural extracellular matrix (ECM), which is an important component of the tissue microenvironment. A cell-free, tissue-specific matrix obtained from pancreas decellularization seems to be the most suitable for creation of a TEPC. The choice of pancreatic tissue decellularization protocol should take into account the morphological characteristics of the original pancreas. Preservation of the architectonics and composition of the native tissue in the decellularized pancreas matrix (DPM), and the presence of native ECM components allow for creation of conditions for prolonged vital activity of functionally active islet (insulin-producing) cells when creating TEPC.Objective: to determine the optimal parameters for decellularization of deceased donor pancreas with fibrosis, lipomatosis, and without pronounced signs of fibrosis and lipomatosis.Materials and methods. We used the caudal part of the pancreas obtained after multiorgan procurement from deceased donors, which was unsuitable for transplantation. Tissue-specific matrix was obtained by a combination of physical and chemical methods of pancreatic decellularization. A freeze-thaw cycle protocol and two protocols using osmotic shock were used. Samples of initial pancreatic tissue and decellularized fragments were subjected to histological analysis.Results. It was shown that a physico-chemical method with freeze-thaw cycles is suitable for effective pancreatic decellularization in severe lipomatosis; a physico-chemical method using osmotic shock, but different protocol variants, is suitable for pancreas with diffuse fibrosis and for pancreas without pronounced signs of fibrosis and lipomatosis.Conclusion. For complete human pancreatic decellularization, the protocol should be correlated with histological features of the original tissue.

Enhancing Peptide Biomaterials for Biofabrication.

Biofabrication using well-matched cell/materials systems provides unprecedented opportunities for dealing with human health issues where disease or injury overtake the body’s native regenerative abilities. Such opportunities can be enhanced through the development of biomaterials with cues that appropriately influence embedded cells into forming functional tissues and organs. In this context, biomaterials’ reliance on rigid biofabrication techniques needs to support the incorporation of a hierarchical mimicry of local and bulk biological cues that mimic the key functional components of native extracellular matrix. Advances in synthetic self-assembling peptide biomaterials promise to produce reproducible mimics of tissue-specific structures and may go some way in overcoming batch inconsistency issues of naturally sourced materials. Recent work in this area has demonstrated biofabrication with self-assembling peptide biomaterials with unique biofabrication technologies to support structural fidelity upon 3D patterning. The use of synthetic self-assembling peptide biomaterials is a growing field that has demonstrated applicability in dermal, intestinal, muscle, cancer and stem cell tissue engineering.

Repopulation of decellularized retinas with hiPSC-derived retinal pigment epithelial and ocular progenitor cells shows cell engraftment, organization and differentiation

The retinal extracellular matrix (ECM) provides architectural support, adhesion and signal guidance that controls retinal development. Decellularization of the ECM affords great potential to tissue engineering; however, how structural retinal ECM affects in vitro development, differentiation and maturation of ocular cells remains to be elucidated. Here, mouse and porcine retinas were decellularized and the protein profile analyzed. Acellular retinal ECM (arECM) scaffolds were then repopulated with human iPSC-derived retinal pigment epithelial (RPE) cells or ocular progenitor cells (OPC) to assess their integration, proliferation and organization. 3837 and 2612 unique proteins were identified in mouse and porcine arECM, respectively, of which 93 and 116 proteins belong to the matrisome. GO analysis shows that matrisome-related proteins were associated with the extracellular region and cell junction and KEGG pathways related to signalling transduction, nervous and endocrine systems and cell junctions were enriched. Interestingly, mouse and porcine arECMs were successfully repopulated with both RPE and OPC, the latter exhibiting cell lineage-specific clusters. Retinal cells organized into different layers containing well-defined areas with pigmented cells, photoreceptors, Müller glia, astrocytes, and ganglion cells, whereas in other areas, conjunctival/limbal, corneal and lens cells re-arranged in cell-specific self-organized areas. In conclusion, our results demonstrated that decellularization of both mouse and porcine retinas retains common native ECM components that upon cell repopulation could guide similar ocular cell adhesion, migration and organization.

Reproducing the Biomechanical Environment of the Chondrocyte for Cartilage Tissue Engineering.

It is well known that the biomechanical and tribological performance of articular cartilage is inextricably linked to its extracellular matrix (ECM) structure and zonal heterogeneity. Furthermore, it is understood that the presence of native ECM components, such as collagen II and aggrecan, promote healthy homeostasis in the resident chondrocytes. What is less frequently discussed is how chondrocyte metabolism is related to the extracellular mechanical environment, at both the macro and microscale. The chondrocyte is in immediate contact with the pericellular matrix of the chondron, which acts as a mechanocoupler, transmitting external applied loads from the ECM to the chondrocyte. Therefore, components of the pericellular matrix also play essential roles in chondrocyte mechanotransduction and metabolism. Recreating the biomechanical environment through tuning material properties of a scaffold and/or the use of external cyclic loading can induce biosynthetic responses in chondrocytes. Decellularized scaffolds, which retain the native tissue macro- and microstructure also represent an effective means of recapitulating such an environment. The use of such techniques in tissue engineering applications can ensure the regeneration of skeletally mature articular cartilage with appropriate biomechanical and tribological properties to restore joint function. Despite the pivotal role in graft maturation and performance, biomechanical and tribological properties of such interventions is often underrepresented. This review outlines the role of biomechanics in relation to native cartilage performance and chondrocyte metabolism, and how application of this theory can enhance the future development and successful translation of biomechanically relevant tissue engineering interventions. Impact statement Physiological cartilage function is a key criterion in the success of a cartilage tissue engineering solution. The in situ performance is dependent on the initial scaffold design as well as extracellular matrix deposition by endogenous or exogenous cells. Both biological and biomechanical stimuli serve as key regulators of cartilage homeostasis and maturation of the resulting tissue-engineered graft. An improved understanding of the influence of biomechanics on cellular function and consideration of the final biomechanical and tribological performance will help in the successful development and translation of tissue-engineered grafts to restore natural joint function postcartilage trauma or osteoarthritic degeneration, delaying the requirement for prosthetic intervention.

Using Acoustic Fields to Fabricate ECM-Based Biomaterials for Regenerative Medicine Applications.

Ultrasound is emerging as a promising tool for both characterizing and fabricating engineered biomaterials. Ultrasound-based technologies offer a diverse toolbox with outstanding capacity for optimization and customization within a variety of therapeutic contexts, including improved extracellular matrix-based materials for regenerative medicine applications. Non-invasive ultrasound fabrication tools include the use of thermal and mechanical effects of acoustic waves to modify the structure and function of extracellular matrix scaffolds both directly, and indirectly via biochemical and cellular mediators. Materials derived from components of native extracellular matrix are an essential component of engineered biomaterials designed to stimulate cell and tissue functions and repair or replace injured tissues. Thus, continued investigations into biological and acoustic mechanisms by which ultrasound can be used to manipulate extracellular matrix components within three-dimensional hydrogels hold much potential to enable the production of improved biomaterials for clinical and research applications.

Cell culture of differentiated human salivary epithelial cells in a serum-free and scalable suspension system: The salivary functional units model.

Saliva aids in digestion, lubrication, and protection of the oral cavity against dental caries and oropharyngeal infections. Reduced salivary secretion, below an adequate level to sustain normal oral functions, is unfortunately experienced by head and neck cancer patients treated with radiotherapy and by patients with Sjögren's syndrome. No disease-modifying therapies exist to date to address salivary gland hypofunction (xerostomia, dry mouth) because pharmacotherapies are limited by the need for residual secretory acinar cells, which are lost at the time of diagnosis, whereas novel platforms such as cell therapies are yet immature for clinical applications. Autologous salivary gland primary cells have clinical utility as personalized cell therapies, if they could be cultured to a therapeutically useful mass while maintaining their in vivo phenotype. Here, we devised a serum-free scalable suspension culture system that grows partially digested human salivary tissue filtrates composing of acinar and ductal cells attached to their native extracellular matrix components while retaining their 3D in vivo spatial organization; we have coined these salivary spheroids as salivary functional units (SFU). The proposed SFU culture system was sub-optimal, but we have found that the cells could still survive and grow into larger salivary spheroids through cell proliferation and aggregation for 5 to 10days within the oxygen diffusion rates in vitro. In summary, by using a less disruptive cell isolation procedure as the starting point for primary cell culture of human salivary epithelial cells, we demonstrated that aggregates of cells remained proliferative and continued to express acinar and ductal cell-specific markers.

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

Regeneration of a Bioengineered Thyroid Using Decellularized Thyroid Matrix.

Hypothyroidism is a common hormone deficiency condition. Regenerative medicine approaches, such as a bioengineered thyroid, have been proposed as potential therapeutic alternatives for patients with hypothyroidism. This study demonstrates a novel approach to generate thyroid grafts using decellularized rat thyroid matrix. Isolated rat thyroid glands were perfused with 1% sodium dodecyl sulfate to generate a decellularized thyroid scaffold. The rat thyroid scaffold was then recellularized with rat thyroid cell line to reconstruct the thyroid by perfusion seeding technique. As a pilot study, the decellularized rat thyroid scaffold was perfused with human-derived thyrocytes and parathyroid cells. The decellularization process retained the intricate three-dimensional microarchitecture with a perfusable vascular network and native extracellular matrix components, allowing efficient reseeding of the thyroid matrix with the FRTL-5 rat thyroid cell line generating three-dimensional follicular structures in vitro. In addition, the recellularized thyroid showed successful cellular engraftment and thyroid-specific function, including synthesis of thyroglobulin and thyroid peroxidase. Moreover, the decellularized rat thyroid scaffold could further be recellularized with human-derived thyroid cells and parathyroid cells to reconstruct a humanized bioartificial endocrine organ, which maintained expression of critical genes such as thyroglobulin, thyroid peroxidase, and parathyroid hormone. These findings demonstrate the utility of a decellularized thyroid extracellular matrix scaffold system for the development of functional, bioengineered thyroid tissue, which could potentially be used to treat hypothyroidism.

Advances and Biomedical Applications of Polypeptide Hydrogels Derived from α-Amino Acid N-Carboxyanhydride (NCA) Polymerizations.

Polypeptide hydrogels, having the ability to mimic certain properties of natural, native extracellular matrix components, are being actively designed and described for various applications in the construction of tissue engineering scaffolds, living cell encapsulation, and drug delivery systems. Compared to conventional hydrogels, polypeptide hydrogels possess biocompatibility, biodegradability, bioactivity, functional diversity, and structural advantage based on the unique secondary structures (α-helix and β-sheet). Furthermore, the progresses in functional N-carboxyanhydride polymerization combined with advanced orthogonal conjugation techniques significantly promote the development of the polypeptide materials. This progress report focuses on the recent advances in designing and engineering polypeptide hydrogels obtained from ring opening polymerization, highlighting the precise manipulation of their properties for biomedical applications.

Perfusion decellularization of a human limb: A novel platform for composite tissue engineering and reconstructive surgery.

Muscle and fasciocutaneous flaps taken from autologous donor sites are currently the most utilized approach for trauma repair, accounting annually for 4.5 million procedures in the US alone. However, the donor tissue size is limited and the complications related to these surgical techniques lead to morbidities, often involving the donor sites. Alternatively, recent reports indicated that extracellular matrix (ECM) scaffolds boost the regenerative potential of the injured site, as shown in a small cohort of volumetric muscle loss patients. Perfusion decellularization is a bioengineering technology that allows the generation of clinical-scale ECM scaffolds with preserved complex architecture and with an intact vascular template, from a variety of donor organs and tissues. We recently reported that this technology is amenable to generate full composite tissue scaffolds from rat and non-human primate limbs. Translating this platform to human extremities could substantially benefit soft tissue and volumetric muscle loss patients providing tissue- and species-specific grafts. In this proof-of-concept study, we show the successful generation a large-scale, acellular composite tissue scaffold from a full cadaveric human upper extremity. This construct retained its morphological architecture and perfusable vascular conduits. Histological and biochemical validation confirmed the successful removal of nuclear and cellular components, and highlighted the preservation of the native extracellular matrix components. Our results indicate that perfusion decellularization can be applied to produce human composite tissue acellular scaffolds. With its preserved structure and vascular template, these biocompatible constructs, could have significant advantages over the currently implanted matrices by means of nutrient distribution, size-scalability and immunological response.

Coming to terms with tissue engineering and regenerative medicine in the lung.

Lung diseases such as emphysema, interstitial fibrosis, and pulmonary vascular diseases cause significant morbidity and mortality, but despite substantial mechanistic understanding, clinical management options for them are limited, with lung transplantation being implemented at end stages. However, limited donor lung availability, graft rejection, and long-term problems after transplantation are major hurdles to lung transplantation being a panacea. Bioengineering the lung is an exciting and emerging solution that has the ultimate aim of generating lung tissues and organs for transplantation. In this article we capture and review the current state of the art in lung bioengineering, from the multimodal approaches, to creating anatomically appropriate lung scaffolds that can be recellularized to eventually yield functioning, transplant-ready lungs. Strategies for decellularizing mammalian lungs to create scaffolds with native extracellular matrix components vs. de novo generation of scaffolds using biocompatible materials are discussed. Strengths vs. limitations of recellularization using different cell types of various pluripotency such as embryonic, mesenchymal, and induced pluripotent stem cells are highlighted. Current hurdles to guide future research toward achieving the clinical goal of transplantation of a bioengineered lung are discussed.

Hydrogels for lung tissue engineering: Biomechanical properties of thin collagen–elastin constructs

In this study, collagen–elastin constructs were prepared with the aim of producing a material capable of mimicking the mechanical properties of a single alveolar wall. Collagen has been used in a wide range of tissue engineering applications; however, due to its low mechanical properties its use is limited to non load-bearing applications without further manipulation using methods such as cross-linking or mechanical compression. Here, it was hypothesised that the addition of soluble elastin to a collagen hydrogel could improve its mechanical properties. Hydrogels made from collagen only and collagen plus varying amounts elastin were prepared. Young׳s modulus of each membrane was measured using the combination of a non-destructive indentation and a theoretical model previously described. An increase in Young׳s modulus was observed with increasing concentration of elastin. The use of non-destructive indentation allowed for online monitoring of the elastic moduli of cell-seeded constructs over 8 days. The addition of lung fibroblasts into the membrane increased the stiffness of the hydrogels further and cell-seeded collagen hydrogels were found to have a stiffness equal to the theoretical value for a single alveolar wall (≈5kPa). Through provision of some of the native extracellular matrix components of the lung parenchyma these scaffolds may be able to provide an initial building block toward the regeneration of new functional lung tissue.

Vascular Extracellular Matrix in Atherosclerosis

The extracellular matrix (ECM) is an essential component of the human body that is responsible for the proper function of various organs. Changes in the ECM have been implicated in the pathogenesis of several cardiovascular conditions including atherosclerosis, restenosis, and heart failure. Matrix components, such as collagens and noncollagenous proteins, influence the function and activity of vascular cells, particularly vascular smooth muscle cells and macrophages. Matrix proteins have been shown to be implicated in the development of atherosclerotic complications, such as plaque rupture, aneurysm formation, and calcification. ECM proteins control ECM remodeling through feedback signaling to matrix metalloproteinases (MMPs), which are the key players of ECM remodeling in both normal and pathological conditions. The production of MMPs is closely related to the development of an inflammatory response and is subjected to significant changes at different stages of atherosclerosis. Indeed, blood levels of circulating MMPs may be useful for the assessment of the inflammatory activity in atherosclerosis and the prediction of cardiovascular risk. The availability of a wide variety of low-molecular MMP inhibitors that can be conjugated with various labels provides a good perspective for specific targeting of MMPs and implementation of imaging techniques to visualize MMP activity in atherosclerotic plaques and, most interestingly, to monitor responses to antiatheroslerosis therapies. Finally, because of the crucial role of ECM in cardiovascular repair, the regenerative potential of ECM could be successfully used in constructing engineered scaffolds and vessels that mimic properties of the natural ECM and consist of the native ECM components or composite biomaterials. These scaffolds possess a great promise in vascular tissue engineering.

In situgelling polysaccharide‐based hydrogel for cell and drug delivery in tissue engineering

ABSTRACTInjectable hydrogels have attracted a great deal of attention as cell carriers and bioactive agents in regenerative medicine due to their ability to fill complex three‐dimensional (3D) tissue gaps and relative ease ofin vivoadministration. Polysaccharide‐based hydrogels can provide microenvironments that favor tissue regeneration and biocompatibility due to their chemical similarities with native extracellular matrix components. This manuscript reports thein vitroapplication of an injectable chitosan‐based polysaccharide hydrogel for cell and protein delivery. Crosslinked hydrogels were produced by the reaction between the amino functionality of chitosan and the aldehyde of dextran aldehyde resulting in an imine bond (Schiff's base) formation in aqueous solutions. This approach eliminated the use of additional crosslinking agents which may pose undesired side effects regarding cytotoxicity and biocompatibility. Additionally, we demonstrate versatility of the gel in terms of its fabrication, and ability to alter mechanical properties by changing the crosslinking extent due to aldehyde content. Bovine serum albumin (BSA), used as a model protein, followed a steady release pattern from the gel. BSA release was dependent on the extent of hydrogel crosslinking. Increase in crosslinking extent resulted in improved mechanical properties and sustained release of BSA. Human fetal osteoblasts encapsulated into the hydrogel showed at least 70% viability and continued to proliferate underin vitroculture. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci.2014,131, 39934.

Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

Recently, scaffolds for tissue regeneration purposes have been observed to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two differently scaled fibers interspersed evenly throughout the entire construct as well as scaffolds containing fibers composed of two discrete materials, specifically fibrin and poly(ε-caprolactone). In order to accomplish this, multiscale fibrous scaffolds of different compositions were generated using a dual extrusion electrospinning setup with a rotating mandrel. These scaffolds were then characterized for fiber diameter, porosity and pore size and seeded with human mesenchymal stem cells to assess the influence of scaffold architecture and composition on cellular responses as determined by cellularity, histology and glycosaminoglycan (GAG) content. Analysis revealed that nanofibers within a microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration.

Collagen scaffolds derived from fresh water fish origin and their biocompatibility

Collagen, a major component of native extracellular matrix, has diverse biomedical applications. However, its application is limited due to lack of cost-effective production and risk of disease transmission from bovine sources currently utilized. This study describes fabrication and characterization of nano/micro fibrous scaffolds utilizing collagen extracted from fresh water fish origin. This is the first time collagen extracted from fresh water fish origin was studied for their biocompatibility and immunogenicity. The nano/micro fibrous collagen scaffolds were fabricated through self-assembly owing to its amphiphilic nature and were subsequently cross-linked. In vitro degradation study revealed higher stability of the cross-linked scaffolds with only ~50% reduction of mass in 30 days, while the uncross-linked one degraded completely in 4 days. Further, minimal inflammatory response was observed when collagen solution was injected in mice with or without adjuvant, without significant dilution of sera. The fish collagen scaffolds exhibited considerable cell viability and were comparable with that of bovine collagen. SEM and fluorescence microscopic analysis revealed significant proliferation rate of cells on the scaffolds and within 5 days the cells were fully confluent. These findings indicated that fish collagen scaffolds derived from fresh water origin were highly biocompatible in nature.

Nanotechnology in Orthopaedics

Nanotechnology provides a multitude of novel tools for applications in orthopaedics, including the meniscus, osteochondral defects, osseointegration of materials, vertebral disk regeneration and repair, and targeted drug delivery. Meniscus The high incidence of meniscal injuries, coupled with the structure's limited healing capacity, has created a demand for the use of tissueengineered biomaterials in meniscal repair and replacement. Baker et al1 showed that aligned nanofibrous scaffolds formed by electrospinning contain the microstructural features and nanolength scales of native extracellular matrix components and provide a substrate conducive to mesenchymal stem cell (MSC) expression of fibrous chondrogenic markers. Nonetheless, these constructs ultimately failed to achieve mechanical equivalence with fibrochondrocyte controls. However, when a similar MSC-laden scaffold based on poly(ε-caprolactone) was coupled with cyclic, physiologic tensile loading,2 increased fibrocartilage gene expression, collagen deposition, and tensile modulus resulted. Nanotechnology for meniscal repair is still in its infancy, but the initial data appear promising. Osteochondral Defects Injuries to articular cartilage remain one of the most challenging issues in orthopaedics. Current treatments that focus on recruitment of MSCs generally result in mechanically inferior fibrocartilagenous tissue.3 Tissue-engineering strategies to develop scaffolds for osteochondral repair procedures are quite desirable, but to date they have not seen widespread clinical success.4 Design of a multiphase material that incorporates the different biochemical and biomechanical properties of articular cartilage and subchondral bone, and that also can address the independent requirements for cartilage and bone regeneration in an osteochondral defect, is a major challenge. Tampieri et al5 used biomimetic nanotechnology to develop a multiphase gradient scaffold with the biologic and functional properties of both bone and cartilage. An integrated composite was created with a bone-like layer of scaffold, a tidemark region with less mineralization, and a cartilaginous layer; the composite was shown to differentially support cartilage and bone generation in vivo. Promising data from the initial studies of this construct, commercialized as Fin-Ceramica Faenza SpA (Faenza, Italy), led to a recent clinical pilot study for osteochondral defect repair in 28 patients.6 Statistically significant improvement in clinical scores was obtained with a 24-month follow-up, with 70% of patients showing complete filling of the osteochondral defect and complete integration of the graft on MRI. Bone Poor osseointegration can be a major contributor to implant loosening and subsequent failure. Modification of implant surfaces using nanotechnology has great potential for extending the life of the implant. Many nanofiber scaffolds made for tissue engineering have shown increased MSC osteogenic differentiation,7,8 as well as increased osteoblast attachment and proliferation onto nanostructured surfaces, including ceramics and metals.9,10 However, there have been few studies that tested the mechanical strength of the bone-implant interface of nanostructures with increased osseointegration. Bjursten et al11 investigated the influence of nanotopographic surface modification by comparing the bonding strength of two titanium implant surfaces, nanosurfaced titanium dioxide (TiO2) nanotubes and a roughened TiO2 gritblasted surface, in rabbit tibias. The nanostructured titanium showed a ninefold improvement in pull-out strength and greater bone-implant contact and bone formation. Nanocomposites that mimic bone in structure and composition have also been under active investigation. Recently, Zhang and colleagues12,13 developed injectable nanostructured three-dimensional hydrogel scaffolds that showed enhanced osteoblast adhesion and displayed suitable mechanical properties to fill and repair bone defects. Scaffolds with mechanical properties in the range of human cancellous bone have been developed, but fabricating scaffolds with mechanical performance close to compact bone remains a challenge.14 Vertebral Disk Nanotechnology has also been investigated for annulus fibrosus (AF) engineering. Nerurkar and colleagues15,16 created electrospun scaffolds as a template for new AF tissue formation; however, the tensile moduli of these constructs reached only one third to one half that of a native lamella. Subsequently, a bilamellar construct with opposing collagen orientations of ±30° was created that showed a circumferential tensile modulus that closely replicated that of native AF.17 These advances in nanostructured scaffolds for vertebral disk engineering also appear promising. Targeted Drug Therapy Nanotechnology is being used in the field of targeted drug therapy for long-term inhibition of bacterial growth. Although earlier studies successfully incorporated large molecules, such as growth factors, into nanostructured materials,18 more recent studies have created nanofibrous scaffolds that incorporate smaller molecules, such as doxycycline19 and silver particles.20 These can be released in a controlled fashion with long-term duration.19 Recently, Li et al21 developed implant nanocoatings that contain proteins monocyte chemotactic protein-1 and interleukin-12, which prevent infection by enhancement of the recruitment and activation of macrophages. These applications have great potential for use in treatment of dental, periodontal, and bone infections. Within the field of orthopaedics, further investigations into the longterm viability and toxicity of nanomaterials, in addition to direct comparisons of nanostructured materials and traditional materials, will enable a more definitive answer to the promise of this technology.

Nanotechnology in Orthopaedics

Nanotechnology provides a multitude of novel tools for applications in orthopaedics, including the meniscus, osteochondral defects, osseointegration of materials, vertebral disk regeneration and repair, and targeted drug delivery. Meniscus The high incidence of meniscal injuries, coupled with the structure’s limited healing capacity, has created a demand for the use of tissueengineered biomaterials in meniscal repair and replacement. Baker et al 1 showed that aligned nanofibrous scaffolds formed by electrospinning contain the microstructural features and nanolength scales of native extracellular matrix components and provide a substrate conducive to mesenchymal stem cell (MSC) expression of fibrous chondrogenic markers. Nonetheless, these constructs ultimately failed to achieve mechanical equivalence with fibrochondrocyte controls. However, when a similar MSC-laden scaffold based on poly(e-caprolactone) was coupled with cyclic, physiologic tensile loading, 2 increased fibrocartilage gene expression, collagen deposition, and tensile modulus resulted. Nanotechnology for meniscal repair is still in its infancy, but the initial data appear promising.