Search

Photo-cross-linking of tyramine-substituted hyaluronan (HA-Tyr) hydrogels is demonstrated for the first time. HA-Tyr hydrogels are fabricated via a rapid photosensitized process using visible light illumination. Nontoxic conditions offer photoencapsulation of human mesenchymal stromal cells (hMSCs) with high viability. Macroscopic gels can be formed in less than 10 s, and one- and two-photon photopatterning enable 2D and 3D microfabrication. Different degrees of cross-linking induce different swelling/shrinking, allowing for light-induced microactuation. These new tools are complementary to the previously reported horseradish peroxidase/hydrogen peroxide cross-linking and allow sequential cross-linking of HA-Tyr matrices.

BMP-2 and TGF-β1 released from injectable thermoresponsive hydrogels are studied in the presence and absence of branched macromolecules bearing BMP-2 or TGF-β1 affinity binding peptides. The synthesized branched macromolecules and the gelling compositions before and after loading with either BMP-2 or TGF-β1 are characterized physico-chemically and show a significantly lower amount of proteins released in the presence of the affinity binding peptide macromolecules. This study illustrates the potential of affinity binding peptide functionalized dendrimers to modulate the local delivery and availability of growth factors important for musculoskeletal regeneration therapies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Infectious complications occur in a minor but significant portion of the patients undergoing joint replacement surgery or fracture fixation, particularly those with severe open fractures, those undergoing revision arthroplasty or those at elevated risk because of poor health status. Once established, infections are difficult to eradicate, especially in the case of bacterial biofilm formation on implanted hardware. Local antibiotic carriers offer the prospect of controlled delivery of antibiotics directly in target tissues and implant, without inducing toxicity in non-target organs. Polymeric carriers have been developed to optimize the release and targeting of antibiotics. Passive polymeric carriers release antibiotics by diffusion and/or upon degradation, while active polymeric carriers release their antibiotics upon stimuli provided by bacterial pathogens. Additionally, some polymeric carriers gelate in-situ in response to physiological stimuli to form a depot for antibiotic release. As antibiotic resistance has become a major issue, also other anti-infectives such as silver and antimicrobial peptides have been incorporated in research. Currently, several antibiotic loaded biomaterials for local infection prophylaxis are available for use in the clinic. Here we review their advantages and limitations and provide an overview of new materials emerging that may overcome these limitations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The research project entitled "rapid prototyping of custom-made bone-forming tissue engineering constructs" (RAPIDOS) is one of the three unique projects that are the result of the first coordinated call for research proposals in biomaterials launched by the European Union Commission and the National Natural Science Foundation of China in 2013 for facilitating bilateral translational research. We formed the RAPIDOS European and Chinese consortium with the aim of applying technologies creating custom-made tissue engineered constructs made of resorbable polymer and calcium phosphate ceramic composites specifically designed by integrating the following: (1) imaging and information technologies, (2) biomaterials and process engineering, and (3) biological and biomedical engineering for novel and truly translational bone repair solutions. Advanced solid free form fabrication technologies, precise stereolithography, and low-temperature rapid prototyping provide the necessary control to create innovative high-resolution medical implants. The use of Chinese medicine extracts, such as the bone anabolic factor icaritin, which has been shown to promote osteogenic differentiation of stem cells and enhance bone healing in vivo , is a safe and technologically relevant alternative to the intensely debated growth factors delivery strategies. This unique initiative driven by a global consortium is expected to accelerate scientific progress in the important field of biomaterials and to foster strong scientific cooperation between China and Europe.

Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.

Recurrent intervertebral disc (IVD) herniation and degenerative disc disease have been identified as the most important factors contributing to persistent pain and disability after surgical discectomy. An annulus fibrosus (AF) closure device that provides immediate closure of the AF rupture, restores disc height, reduces further disc degeneration and enhances self-repair capacities is an unmet clinical need. In this study, a poly(trimethylene carbonate) (PTMC) scaffold seeded with human bone marrow derived mesenchymal stromal cells (MSCs) and covered with a poly(ester-urethane) (PU) membrane was assessed for AF rupture repair in a bovine organ culture annulotomy model under dynamic load for 14 days. PTMC scaffolds combined with the sutured PU membrane restored disc height of annulotomized discs and prevented herniation of nucleus pulposus (NP) tissue. Implanted MSCs showed an up-regulated gene expression of type V collagen, a potential AF marker, indicating in situ differentiation capability. Furthermore, MSCs delivered within PTMC scaffolds induced an up-regulation of anabolic gene expression and down-regulation of catabolic gene expression in adjacent native disc tissue. In conclusion, the combined biomaterial and cellular approach has the potential to hinder herniation of NP tissue, stabilize disc height, and positively modulate cell phenotype of native disc tissue., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Injectable tyramine modified hyaluronic acid (HA-Tyr) hydrogels which are bio-orthogonally cross-linked with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) are excellent candidate biomaterials for drug delivery, regenerative medicine and tissue engineering. Ligation of tyramine to HA has been reported using the very well established N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) chemistry. Here we demonstrate the applicability of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as an alternative coupling agent to synthesize HA-Tyr conjugates. The optimized derivatization process allows accurate control of the degree of substituted Tyr on hyaluronan (DSmol). Hence, viscoelastic properties, in vitro swelling and enzymatic digestion profiles of the crosslinked hydrogels can be precisely tuned via DSmol. Our study demonstrates the advantages of DMTMM conjugation as a powerful tool to synthesize HA-Tyr hydrogels with properties exactly tailored for biomedical applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Poly(N-isopropylacrylamide) conjugated hyaluronan (HA-pN), a brush-like copolymer system which serves as a polymer vehicle for cellular and drug delivery, has been previously synthesized via the copper catalyzed azide-alkyne reaction (CuAAC) using a combination of copper sulfate and ascorbic acid (CuAsc) as the catalytic system of choice. Bromotris(triphenylphosphine) copper(I) (CuBr(PPh3)3) is an alternative catalytic compound containing a phosphorous ligand which stabilizes copper in the +1 oxidative state in aqueous solvents and can be employed at true catalyst concentrations., Objective: CuAsc and CuBr(PPh3)3 were compared for their efficiency; 1) in the synthesis of HA-pN via CuAAC; 2) in producing thermoresponsive compositions and 3) in being extracted from the polymeric compositions., Methods: The synthesis of the brush copolymer was carried out under strict Schlenk conditions, then characterized by ATR-FTIR, 1H NMR, ICP-SFMS, and rheological measurements., Results: CuBr(PPh3)3 catalyzed CuAAC leads to better grafting in water, at a true catalyst concentration, compared to CuAsc. Polymeric solutions exhibited similar traits of increasing mechanical stiffness with rising temperature. Despite purification via chelation and/or dialysis, residual copper was present in similar concentrations in the final polymers., Conclusions: In the CuAAC driven copolymer synthesis of the HA-pN, CuBr(PPh3)3 is a better catalyst than CuAsc.

Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fragility fractures can cause significant morbidity and mortality in patients with osteoporosis and inflict a considerable medical and socioeconomic burden. Moreover, treatment of an osteoporotic fracture is challenging due to the decreased strength of the surrounding bone and suboptimal healing capacity, predisposing both to fixation failure and non-union. Whereas a systemic osteoporosis treatment acts slowly, local release of osteogenic agents in osteoporotic fracture would act rapidly to increase bone strength and quality, as well as to reduce the bone healing period and prevent development of a problematic non-union. The identification of agents with potential to stimulate bone formation and improve implant fixation strength in osteoporotic bone has raised hope for the fast augmentation of osteoporotic fractures. Stimulation of bone formation by local delivery of growth factors is an approach already in clinical use for the treatment of non-unions, and could be utilized for osteoporotic fractures as well. Small molecules have also gained ground as stable and inexpensive compounds to enhance bone formation and tackle osteoporosis. The aim of this paper is to present the state of the art on local drug delivery in osteoporotic fractures. Advantages, disadvantages and underlying molecular mechanisms of different active species for local bone healing in osteoporotic bone are discussed. This review also identifies promising new candidate molecules and innovative approaches for the local drug delivery in osteoporotic bone., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The combination of biomaterials and drug delivery strategies is a promising avenue towards improved synthetic bone substitutes. With the delivery of active species biomaterials can be provided with the bioactivity they still lack for improved bone regeneration. Recently, a lot of research efforts have been put towards this direction. Biomaterials for bone regeneration have been supplemented with small or biological molecules for improved osteoprogenitor cell recruitment, osteoinductivity, anabolic or angiogenic response, regulation of bone metabolism and others. The scope of this review is to summarize the most recent results in this field.

Adipose tissue-derived microvascular fragments are promising vascularisation units for applications in the field of tissue engineering. Elderly patients are the major future target population of such applications due to an increasing human life expectancy. Therefore, we herein investigated the effect of aging on the fragments' vascularisation capacity. Microvascular fragments were isolated from epididymal fat pads of adult (8 months) and aged (16 months) C57BL/6 donor mice. These fragments were seeded onto porous polyurethane scaffolds, which were implanted into dorsal skinfold chambers to study their vascularisation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with fragments from aged donors exhibited a significantly lower functional microvessel density and intravascular blood flow velocity. This was associated with an impaired vessel maturation, as indicated by vessel wall irregularities, constantly elevated diameters and a lower fraction of CD31/α-smooth muscle actin double positive microvessels in the implants' border and centre zones. Additional in vitro analyses revealed that microvascular fragments from adult and aged donors do not differ in their stem cell content as well as in their release of angiogenic growth factors, survival and proliferative activity under hypoxic conditions. However, fragments from aged donors exhibit a significantly lower number of matrix metalloproteinase -9-positive perivascular cells. Taken together, these findings demonstrate that aging is a crucial determinant for the vascularisation capacity of isolated microvascular fragments.

Undifferentiated adipose-derived mesenchymal stem cell (adMSC) spheroids are attractive vascularization units for tissue engineering. Their osteogenic differentiation further offers the possibility of directed generation of bone constructs. The aim of this study was to analyze how this differentiation affects their in vivo vascularization capacity. Green fluorescent protein (GFP)-positive adMSCs were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of undifferentiated and differentiated spheroids using the liquid overlay technique. Subsequently, polyurethane scaffolds were seeded with these spheroids and successful osteogenic differentiation was proven by von Kossa staining and high-resolution microtomography. The scaffolds were then implanted into dorsal skinfold chambers of C57BL/6 wild-type mice to analyze their vascularization and incorporation using intravital fluorescence microscopy, histology and immunohistochemistry. Scaffolds seeded with differentiated spheroids exhibited a markedly impaired vascularization. Immunohistochemical analyses revealed that this was caused by the lost ability of differentiated spheroids to form GFP-positive microvascular networks inside the scaffolds. This was associated with a reduced tissue incorporation of the implants. Moreover, they no longer exhibited a mineralized matrix after the 14day implantation period, indicating the dedifferentiation of the spheroids under the given in vivo conditions. These findings indicate that osteogenic differentiation of adMSC spheroids markedly impairs their vascularization capacity. Hence, it may be reasonable to combine adMSC spheroids of varying differentiation stages in scaffolds for bone tissue engineering to promote both vascularization and bone formation., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Intervertebral disc (IVD) degeneration is the leading cause of low back pain and disability in the active population. Transplantation of mesenchymal stem cells (MSCs) in a hydrogel carrier can induce regenerative effects in degenerated IVDs. Moreover, it was found that degenerative discs release chemoattractants effective in MSC recruitment. Based on these findings, we hypothesized that an injectable hydrogel that can enhance the number of migrated MSCs in the IVD and provide a suitable matrix for their survival and differentiation would be ideal. The purpose of this study was to evaluate the potential of a thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HAP) hydrogel as chemoattractant delivery system to recruit human MSCs in degenerative IVDs. The results demonstrate that HAP hydrogels containing stromal cell derived factor-1 (SDF-1) significantly increased the number of MSCs migrating into nucleotomized discs compared with discs treated with only HAP or SDF-1 in solution. HAP hydrogels releasing SDF-1 enhanced both the number of recruited cells and their migration distance in the IVD tissue. Furthermore, this phenomenon was dependent on MSC donor age. In conclusion, HAP SDF-1 is effective for the recruitment of stem cells in the IVD, thus opening new possibilities for the development of regenerative therapies based on endogenous cell migration., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The activation of carboxyl groups with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) for amide formation is the standard method for amine ligation to hyaluronan (HA), and a very well established wide-ranging bioconjugation method. In this paper we compare 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) to EDC/NHS activation chemistry for HA ligation using an array of substrates including small, large and functional molecules. For all the substrates tested DMTMM yields were superior at parity of feed ratio. DMTMM chemistry resulted effective also in absence of pH control, which is essential for EDC/NHS conjugation. Overall our results demonstrate that DMTMM is more efficient than EDC/NHS for ligation of amines to HA and does not require accurate pH control or pH shift during the reaction to be effective. DMTMM-mediated ligation is a new promising chemical tool to synthesize HA derivatives for biomedical and pharmaceutical applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The purpose of this study was to evaluate the impact on osteochondral healing of press-fitted multiphasic osteochondral scaffolds consisting of poly(ester-urethane) (PUR) and hydroxyapatite into a cylindric osteochondral defect in the distal non-weight bearing femoral trochlear ridge of the rabbit. Two scaffolds were investigated, one with and one without an intermediate microporous membrane between the cartilage and the bone compartment of the scaffold. A control group without a scaffold placed into the defect was included. After 12 weeks macroscopic and histomorphological analyses were performed. The scaffold was easily press-fitted and provided a stable matrix for tissue repair. The membrane did not demonstrate a detrimental effect on tissue healing compared with the scaffold without membrane. However, the control group had statistically superior healing as reflected by histological differences in the cartilage and subchondral bone compartment between control group and each scaffold group. A more detailed analysis revealed that the difference was localized in the bone compartment healing. The present study demonstrates that an elastomeric PUR scaffold can easily be press-fitted into an osteochondral defect and provides a stable matrix for tissue repair. However, the multi-phasic scaffold did not provide a clear advantage for tissue healing. Future investigations should refine especially the bone phase of the implant to increase its stiffness, biocompatibility and osteoconductive activity. A more precise fabrication technique would be necessary for the matching of tissue organisation.

Machined and injection moulded polyetheretherketone (PEEK) implants with and without an oxygen plasma modification were prepared and implanted in sheep cancellous and cortical bone. After 4, 12 and 26 weeks, osseointegration was evaluated through mechanical push-out tests and histomorphometry. In the cancellous bone, push-out force increased with time, a trend toward higher force was observed for machined compared to moulded, and oxygen plasma modified compared to unmodified. On-going remodelling of the bone was detected in the periphery of the implants at 4 weeks. Minimal or no inflammation was observed with all the implants at all locations and time-points. Bone-implant contact (BIC) was quantified at all-time points and locations for all the four PEEK implant surfaces. The BIC values ranged from 15 to 75% with an average of 29 ± 13% in the cancellous bone and 25-65% with an average of 50 ± 12% in the cortical bone. In the cortical bone the BIC increased significantly from 4 to 26 weeks. This in vivo study has identified that surface topography of PEEK implants influences osseointegration. In addition, oxygen plasma has the potential to increase bone-implant interface stability. This study provides a unique reference for further modifications and in vivo assessment of PEEK implants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hydrogels pose interesting features for cartilage regeneration strategies, such as the option for injectability and in situ gelation resulting in optimal filling of defects. We aimed to study different hydrogels for their capability to support chondrogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs). hBMSCs were encapsulated in alginate, alginate with hyaluronic acid (alginate/HA), fibrin or thermoresponsive HA grafted with poly(N-isopropyl acrylamide) side-chains (HA-pNIPAM). Glycosaminoglycan production and cartilage-related gene expression were significantly higher in hBMSC-alginate and hBMSC-fibrin constructs than in the other constructs. Supplementation of alginate with HA was not beneficial. hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs were placed in simulated defects in osteochondral biopsies and cultured in vitro for 28 d. Biopsies containing hBMSC-alginate and hBMSC-fibrin were implanted subcutaneously in nude mice for 12 weeks. hBMSC-alginate constructs had significantly higher cartilage-related gene expression after 28 d of culture as well as significantly more safranin-O positive repair tissue after 12 weeks in vivo than hBMSC-fibrin constructs. Although initial experiments with hBMSC-hydrogel constructs suggested comparable results of hBMSC-alginate, hBMSC-fibrin and hBMSC-HA-pNIPAM constructs, culture in the osteochondral biopsy model in vitro as well as in vivo revealed differences, suggests that chondrogenesis of hBMSCs in an osteochondral environment is hydrogel-dependent.

Electrospinning technology is an attractive process for the fabrication of a scaffold with an annulus fibrosus (AF)-like architecture for tissue engineering. Oriented and nonoriented electrospun scaffolds were prepared from poly(ester-urethane) (PU) and poly(ɛ-caprolactone) (PCL) as well as corresponding homogeneous films. Scaffolds' characteristics and mechanical properties were characterized by scanning electron microscopy, static water contact measurements, and dynamic mechanical analysis, respectively. The effect of scaffold architecture and polymer composition on bovine AF cells was investigated. PU and PCL films and scaffolds supported AF cell growth and extracellular matrix production and accumulation. Electrospun scaffolds increased the retention of collagen and glycosaminoglycan compared with films. Fiber orientation of the scaffolds promoted the AF cell phenotype with a trend toward an upregulation of matrix gene expression for oriented relative to nonoriented scaffolds. The higher yield strain of an oriented electrospun PU scaffold, compared with other scaffolds, will be advantageous for AF tissue engineering under a dynamic mechanical environment.

Background Context: The fate of human mesenchymal stem cells (hMSCs) supplied to the degenerating intervertebral disc (IVD) is still not fully understood and can be negatively affected by low oxygen, pH, and glucose concentration of the IVD environment. The hMSC survival and yield upon injection of compromised IVD could be improved by the use of an appropriate carrier and/or by predifferentiation of hMSCs before injection., Purpose: To optimize hMSC culture conditions in thermoreversible hyaluronan-based hydrogel, hyaluronan-poly(N-isopropylacrylamide) (HA-pNIPAM), to achieve differentiation toward the disc phenotype in vitro, and evaluate whether preconditioning contributes to a better hMSC response ex vivo., Study Design: In vitro and ex vivo whole-organ culture of hMSCs., Methods: In vitro cultures of hMSCs were conducted in HA-pNIPAM and alginate for 1 week under hypoxia in chondropermissive medium alone and with the supplementation of transforming growth factor β1 or growth and differentiation factor 5 (GDF-5). Ex vivo, hMSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or predifferentiated with GDF-5 for 1 week in HA-pNIPAM and then supplied to the IVDs. Cell viability was evaluated by Live-Dead assay, and DNA, glycosaminoglycan (GAG), and gene expression profiles were used to assess hMSC differentiation toward the disc phenotype., Results: The HA-pNIPAM induced hMSC differentiation toward the disc phenotype more effectively than alginate: in vitro, higher GAG/DNA ratio and higher collagen type II, SOX9, cytokeratin-19, cluster of differentiation 24, and forkhead box protein F1 expressions were found for hMSCs cultured in HA-pNIPAM compared with those cultured in alginate, regardless of the addition of growth factors. Ex vivo, direct combination of HA-pNIPAM with the disc environment induced a stronger disc-like differentiation of hMSCs than predifferentiation of hMSCs followed by their delivery to the discs., Conclusions: Hyaluronan-based thermoreversible hydrogel supports hMSC differentiation toward the disc phenotype without the need for growth factor supplementation in vitro and ex vivo. Further in vivo studies are required to confirm the suitability of this hydrogel as an effective stem cell carrier for the treatment of IVD degeneration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach - combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.

The combination of hydrogels and calcium phosphate particles is emerging as a well-established trend for bone substitutes. Besides acting as binders for the inorganic phase, hydrogels within these hybrid materials can modulate cell colonization physically and biologically. The influence of hydrogels on the healing process can also be exploited through their capability to deliver drugs and cells for tissue engineering approaches. The aim of this review is to collect some recent progress in this field, with an emphasis on design aspects and possible future directions., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Lumbar discectomy is the surgical procedure most frequently performed for patients suffering from low back pain and sciatica. Disc herniation as a consequence of degenerative or traumatic processes is commonly encountered as the underlying cause for the painful condition. While discectomy provides favourable outcome in a majority of cases, there are conditions where unmet requirements exist in terms of treatment, such as large disc protrusions with minimal disc degeneration; in these cases, the high rate of recurrent disc herniation after discectomy is a prevalent problem. An effective biological annular repair could improve the surgical outcome in patients with contained disc herniations but otherwise minor degenerative changes. An attractive approach is a tissue-engineered implant that will enable/stimulate the repair of the ruptured annulus. The strategy is to develop three-dimensional scaffolds and activate them by seeding cells or by incorporating molecular signals that enable new matrix synthesis at the defect site, while the biomaterial provides immediate closure of the defect and maintains the mechanical properties of the disc. This review is structured into (1) introduction, (2) clinical problems, current treatment options and needs, (3) biomechanical demands, (4) cellular and extracellular components, (5) biomaterials for delivery, scaffolding and support, (6) pre-clinical models for evaluation of newly developed cell- and material-based therapies, and (7) conclusions. This article highlights that an interdisciplinary approach is necessary for successful development of new clinical methods for annulus fibrosus repair. This will benefit from a close collaboration between research groups with expertise in all areas addressed in this review.

Specific bacteria imaging is highly desirable in clinical diagnostics. Probes enabling rapid and specific diagnostics of bacteria are limited. Current clinical infection diagnostics is time consuming and invasive, relies on microbiological cultures. We investigated the potential of Lysostaphin as a specific probe to label staphylococci in a new labeling protocol. We used azido (N(3))-modified Lysostaphin-N(3) and DIBO-dye in a two-step bacteria-labeling protocol. N(3) and DIBO (di-benzocyclooctyne) are the counterparts of the "click" chemistry. In the first step, Lysostaphin-N(3) binds specifically to Staphylococcus aureus. In the second step, N(3) clicks to DIBO thus achieving the selective for S. aureus labeling. Such a two-step approach effectively distinguishes S. aureus from Escherichia coli; non-toxic and was proven to work in vivo. The two-step labeling protocol is a promising approach for diagnostic imaging of staphylococcal infections in clinical settings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An efficient and scale-up ready single-step synthesis for the conjugation of thermoresponsive polymers to hyaluronic acid (HA) was established. Jeffamines(®) (JFM) and poly(N-isopropylacrylamide) (PNIPAM) were grafted to HA via direct amidation mediated by 1,1'-carbonyldiimidazole activation. The temperature-induced gelation of the semi-synthetic co-polymers was characterized by rheology as a function of the temperature and by differential scanning calorimetry (DSC). A HA-JFM conjugate with sol-gel transition in a physiologically relevant temperature range was identified. The grafting of PNIPAM resulted in the drastic change of the main rheological properties of native HA, revealing the hydrophobic non-covalent nature of the interactions between the thermoresponsive brushes in the gel state. Owing to the reversibility of these interactions and the sharpness of the transition, the HA-PNIPAM conjugates are suitable candidates for the incorporation of drugs, cells or ceramic materials for different biomedical applications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In tissue engineering, the generation of tissue constructs comprising preformed microvessels is a promising strategy to guarantee their adequate vascularisation after implantation. Herein, we analysed whether this may be achieved by seeding porous scaffolds with adipose tissue-derived microvascular fragments. Green fluorescent protein (GFP)-positive microvascular fragments were isolated by enzymatic digestion from epididymal fat pads of male C57BL/6-TgN(ACTB-EGFP)1Osb/J mice. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were seeded with these fragments and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study inosculation and vascularisation of the implants by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Empty scaffolds served as controls. Vital microvascular fragments could be isolated from adipose tissue and seeded onto the scaffolds under dynamic pressure conditions. In the dorsal skinfold chamber, the fragments survived and exhibited a high angiogenic activity, resulting in the formation of GFP-positive microvascular networks within the implants. These networks developed interconnections to the host microvasculature, resulting in a significantly increased functional microvessel density at day 10 and 14 after implantation when compared to controls. Immunohistochemical analyses of vessel-seeded scaffolds revealed that >90 % of the microvessels in the implants' centre and ~60 % of microvessels in the surrounding host tissue were GFP-positive. This indicates that the scaffolds primarily vascularised by external inosculation. These novel findings demonstrate that the vascularisation of implanted porous scaffolds can be improved by incorporation of microvascular fragments. Accordingly, this approach may markedly contribute to the success of future tissue engineering applications in clinical practice.

Introduction: Thermoreversible hydrogels have potential in spine research as they provide easy injectability and mild gelling mechanism (by physical cross-link). The purpose of this study was to assess the potential of thermoreversible hyaluronan-based hydrogels (HA-pNIPAM) (pNIPAM Mn = 10, 20, 35 × 10(3) g mol(-1)) as nucleus pulposus cells (NPC) carrier., Materials and Methods: Cytocompatibility (WST-1 assay), viability (trypan blue), morphology (toluidine blue), sulphated glycosaminoglycan synthesis (DMMB assay) and gene expression profile (real-time PCR) of bovine NPC cultured in HA-pNIPAM were followed for 1 week and compared to alginate gel bead cultures. The injectability and cell survival in a whole disc organ culture model were assessed up to day 7., Results: All HA, HA-pNIPAM and their degradation products were cytocompatible to NPC. HA-pNIPAM hydrogels with no volume change upon gelling maintained NPC viability and characteristic rounded morphology. Glycosaminoglycan synthesis was similar in HA-pNIPAM and alginate gels. Following NPC expansion, both gels induced re-differentiation toward the NPC phenotype. Significant differences between the two gels were found for COLI, COLII, HAS1, HAS2 and ADAMTS4 but not for MMPs and TIMPs. Higher expression of hyaluronan synthases (HAS1, HAS2) and lower expression of COLI and COLII mRNA were noted in cells cultured in HA-pNIPAM (pNIPAM = 20 × 10(3)g mol(-1)). NPC suspension in HA-pNIPAM was injectable through a 22-G needle without loss of cell viability. Ex vivo, NPC viability was maintained in HA-pNIPAM for 1 week., Conclusion: A HA-pNIPAM composition suitable for nucleus pulposus repair that provides an injectable carrier for NPC, maintains their phenotype and promotes extracellular matrix generation was identified.

Inosculation of preformed microvessels with the host microvasculature represents a promising approach to accelerate vascularization of tissue constructs. Herein, we analyzed whether cultivation of prevascularized tissue constructs promotes inosculation by reducing the perivascular cell coverage of the preformed microvessels. Poly(ester-urethane) scaffolds were implanted into FVB/N-TgN (Tie2/green fluorescent protein [GFP]) 287 Sato mice to generate prevascularized tissue constructs with GFP-positive microvessels. These constructs were then cultivated for 3 or 10 days before implantation into dorsal skinfold chambers of FVB/N mice to analyze inosculation and vascularization by intravital fluorescence microscopy and immunohistochemistry. Noncultivated tissue constructs served as controls. Cultivation reduced the number of α-smooth muscle actin-positive preformed microvessels within the constructs and increased the production of vascular endothelial growth factor. After 3 days of cultivation, tissue constructs still exhibited good cell viability, whereas apoptotic cell death was massively increased in the 10-day-cultivated group. After implantation, inosculation of preformed microvessels was accelerated in the 3-day-cultivated constructs. This resulted in an improved vascularization, as indicated by an increased functional microvessel density and blood perfusion. Immunohistochemical detection of GFP-positive microvessels revealed that internal and external inosculation occurs in parallel. In conclusion, this study demonstrates that inosculation of in situ prevascularized tissue constructs can be easily accelerated by destabilization of preformed microvessels and angiogenic activation during short-term cultivation.

The engineering of preformed microvessels offers the promising opportunity to rapidly vascularise implanted tissue constructs by the process of inosculation. Herein, we analyzed whether this process may further be accelerated by cultivation of prevascularised tissue constructs in Matrigel before implantation. Nano-size hydroxyapatite particles/poly(ester-urethane) scaffolds were implanted into the flank of FVB/N-TgN (Tie2/GFP) 287 Sato mice to allow the ingrowth of a granulation tissue with green fluorescent protein (GFP)-positive blood vessels. After harvesting, these prevascularised constructs were then transferred into dorsal skinfold chambers of FVB/N recipient mice to study the process of inosculation. The constructs were implanted directly after embedding in Matrigel or after 3 days of cultivation in the extracellular matrix. Matrigel-free constructs served as control. Cultivation in Matrigel resulted in the outgrowth of CD31/GFP-positive vascular sprouts. Vascularisation of these constructs was markedly improved when compared to the other two groups, as indicated by a significantly elevated functional microvessel density between days 6 to 14 after implantation into the dorsal skinfold chamber. This was associated with an increased number of GFP-positive blood vessels growing into the surrounding host tissue. Thus, the blood supply to prevascularised tissue constructs can be accelerated by their pre-cultivation in an angiogenic extracellular matrix, promoting external inosculation of the preformed microvascular networks with the host microvasculature.

Thermoreversible hydrogels are promising matrices for tissue-engineered cartilage and spine constructs. They require specific properties during all the stages of a cell therapy (e.g., cell expansion, recovery, injection, delivery). Thermoreversible hyaluronan-poly(N-isopropylacrylamide) (HA-PNIPAM) hydrogels with well-defined molecular architecture and properties were synthesized through RAFT polymerization and "click" chemistry. The effect of PNIPAM grafting length and density on HA-PNIPAM properties was evaluated by methods relevant for a cell therapy. It was found that reversibility of the PNIPAM gelling process was improved in the presence of HA. Increasing M(n) of PNIPAM decreased the viscosity at 20 degrees C and led to high G' at T > 30 degrees C; however, higher grafting density led to lower mechanical properties. Water uptake of the hydrogels was mainly dependent on PNIPAM M(n). All of the hydrogels and their degradation products were cytocompatible to hTERT-BJ1 fibroblasts. A composition with properties ideal for cell encapsulation was identified and characterized by a low viscosity at 20 degrees C, rapid gelling at 37 degrees C, absence of volume change upon gelling, and G' of 140 Pa at 37 degrees C.

A bifunctionalized 3,7,11-trimethyl-2,6,10-dodecatrien-1-diaminobutane amide (isoprenoid) was obtained from 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol (farnesol) in a three-step synthesis. The bifunctionalized isoprenoid was characterized using infrared spectroscopy and (1)H and (13)C nuclear magnetic resonance spectroscopy and was covalently incorporated (0.12 mmol x g(-1)) into the biodegradable aliphatic polyurethane formed on the polycondensation reaction of poly(epsilon-caprolactone) diol, 1,4,3,6-dianhydro-D-sorbitol and 1,6-hexamethylene diisocyanate. Although the covalent incorporation of the isoprenoid molecule into the polyurethane chain modified the surface chemistry of the polymer, it did not affect the viability of attached chondrocytes. Porous 3D scaffolds were produced from the modified and unmodified biodegradable segmented polyurethanes by a salt leaching-phase-inverse technique. The scaffolds were seeded with bovine chondrocytes encapsulated in fibrin gel and cultured in vitro for 14 days. The incorporation of bifunctional isoprenoid into the polyurethane affected the morphology of the scaffolds produced, when compared with the morphology of the scaffolds produced using the same technique from the unmodified polyurethane. As a consequence, there was more uniform cell seeding and more homogeneous distribution of the synthesized extracellular matrix throughout the scaffold resulting in a reduced cell/tissue layer at the edges of the constructs. However, glycosaminoglycan (GAG), DNA content, and chondrocytes phenotype in the scaffolds produced from these two polyurethane formulations did not vary significantly. The findings suggest that the change of surface characteristics and the more open pore structure of the scaffolds produced from the isoprenoid-modified polyurethane are beneficial for the seeding efficiency and the homogeneity of the tissue engineered constructs.

Poly(thiourethane-urethane)s with varying amounts of sulphur were synthesised by a two-step polycondensation consisting of the sequential addition of 1,6-hexamethylene diisocyanate and bis(2-mercaptoethyl) ether in a poly(epsilon-caprolactone) diol solution. Polymers prepared had high weight-average molecular weight and typical microdomains separation, as shown by size-exclusion chromatography and thermal analysis. Polymer surfaces were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. The quantification of thiol groups at the surface was assessed using a fluorescent assay. Thiol concentration ranged between 7 and 14 nmol/cm, and was directly related to the amount of sulphur introduced in the polymerization and the macromolecule chains orientation at the surfaces. A preliminary in vitro degradation study and a proliferation assay were performed. The poly(thiourethane-urethane)s may have important applications as biodegradable and biocompatible materials for cartilage and bone tissue engineering. The surface thiol groups add the prospect of further functionalization. This is an important benefit compared to biodegradable poly(urethane)s that usually present low biological activity.

Scaffolds for tissue engineering should be biocompatible and stimulate rapid blood vessel ingrowth. Herein, we analyzed in vivo the biocompatibility and vascularization of three novel types of biodegradable porous polyurethane scaffolds. The polyurethane scaffolds, i.e., PU-S, PU-M and PU-F, were implanted into dorsal skinfold chambers of BALB/c mice. Using intravital fluorescence microscopy we analyzed vascularization of the implants and venular leukocyte-endothelial cell interaction in the surrounding host tissue over a 14 day period. Incorporation of the scaffolds was analyzed by histology, and a WST-1 assay was performed to evaluate their cell biocompatibility in vitro. Our results indicate that none of the polyurethane scaffolds was cytotoxic. Accordingly, rolling and adherent leukocytes in venules of the dorsal skinfold chamber were found in a physiological range after scaffold implantation and did not significantly differ between the groups, indicating a good in vivo biocompatibility. However, the three scaffolds induced a weak angiogenic response with a microvessel density of only approximately 47-60 and approximately 3-10cm/cm(2) in the border and centre zones of the scaffolds at day 14 after implantation. Histology demonstrated that the scaffolds were incorporated in a granulation tissue, which exhibited only a few blood vessels and inflammatory cells. In conclusion, PU-S, PU-M and PU-F scaffolds may be used to generate tissue constructs which do not induce a strong inflammatory reaction after implantation into patients. However, the scaffolds should be further modified or conditioned in order to accelerate and improve the process of vascularization.

Cartilage defects are notoriously difficult to repair and owing to the long-term prognosis of osteoarthritis, and a rapidly aging population, a need for new therapies is pressing. Cell-based therapies for cartilage regeneration were introduced into patients in the early 1990s. Since that time the technology has developed from a simple cell suspension to more complex 3D structures. Cells, both chondrocytes and stem cells, have been incorporated into scaffold material with the aim to better recreate the natural environment of the cell, while providing more structural support to withstand the large forces applied on the de novo tissue. This review aims to provide an overview of potential cell sources and different scaffold materials, which are in development for cartilage tissue engineering.

This report summarizes the state of the art and recent developments and advances in the use of degradable polymers devices for osteosynthesis. The current generation of biodegradable polymeric implants for bone repair utilising designs copied from metal implants, originates from the concept that devices should be supportive and as "inert" substitute to bone tissue. Today degradable polymeric devices for osteosynthesis are successful in low or mild load bearing applications. However, the lack of carefully controlled randomized prospective trials that document their efficacy in treating a particular fracture pattern is still an issue. Then, the choice between degradable and non-degradable devices must be carefully weighed and depends on many factors such as the patient age and condition, the type of fracture, the risk of infection, etc. The improvement of the biodegradable devices mechanical properties and their degradation behaviour will have to be achieved to broaden their use. The next generation of biodegradable implants will probably see the implementation of the recent gained knowledge in cell-material interactions and cells therapy, with a better control of the spatial and temporal interfaces between the material and the surrounding bone tissue.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

This review concerns the importance of length and time on physicochemical interactions between living tissue and biomaterials that occur on implantation. The review provides information on material host interactions, materials for medical applications and cell surface interactions, and then details the extent of knowledge concerning the role(s) that surface chemistry and topography play during the first stage of implant integration, namely protein adsorption. The key points are illustrated by data from model in vitro studies. Host implant interactions begin nanoseconds after first contact and from then on are in a state of flux due to protein adsorption, cell adhesion and physical and chemical alteration of the implanted material. The many questions concerning the conformational form and control of bound proteins and how this may impact on cell adhesion in the first instance and later on cell signalling and implant integration can be answered by systematic investigations using model materials. Only then we will be in a more informed position to design new materials for use in the body.

Type I collagen hydrogel containing bioactive glass (CaO-P2O5-SiO2) and silica sol-gel micrometric particles were prepared and their in vitroapatite-forming ability in simulated body fluid assessed. X-ray diffraction and scanning electron microscopy analysis showed that bioactive glass particles entrapment in collagen matrix did not inhibit calcium phosphate formation and induced morphology variations on the crystalline phase precipitated on the hydrogel surface. The silica--collagen hydrogel composite precipitated calcium phosphate whereas silica particles and collagen hydrogel alone did not, indicating a possible synergetic effect between collagen and silica on the apatite-forming ability. Mechanisms of calcium phosphate precipitation and its relevance to biomaterial development are discussed.

Type I collagen is a suitable and versatile template for the structuration of silica at different length scales.

Collagen-silica hybrid materials have been considered for potential biomedical applications. Understanding of the collagen-silica interactions is the key to control hybrids structure and properties. For this purpose, the effect of sodium silicate and sodium chloride addition at two concentrations, 0.83 and 10 mM, on the kinetic of the type I collagen fibrillogenesis at 20 degrees C, and pH 7.4 were studied. Absorbance profiles of fibrillogenesis experiments were collected together with measures of silicic acid concentration and transmission electron microscopy analysis. The specific effect of silica addition on the collagen fibrils self-assembly mechanisms was demonstrated by comparison with the sodium chloride. Sodium silicate at 10 mM inhibited the collagen fibrillogenesis. At the same concentration, the sodium chloride decreased the rate of the collagen fibril assembly. Collagen fibrillogenesis kinetic was not significantly disturbed by the presence of 0.83 mM of sodium chloride. However, the same concentration of sodium silicate modified the collagen fibrillogenesis kinetic. Transmission electron microscopy indicated for experiment with 0.83 mM of sodium silicate, the formation of longer and wider fibrils than for the equivalent collagen fibrillogenesis experiment with sodium chloride. The effect of sodium chloride is explained in terms of osmotic exclusion and influence on electrostatic interactions between collagen fibrils. The specific involvement of silicic acid in collagen helices hydrogen-bond interactions is suggested. Finally, the results of this study are discussed regarding the preparation of composites by co-gelation of type I collagen and sodium silicate, for potential application as bone repair device.

Skeletal tissues associate in close interaction, a dense organic matrix and a mineral network. In bone, the major structural protein is type I collagen, associated with inorganic crystals of hydroxyapatite. The three-dimensional arrangement of collagen fibrils in compact bone forms regularly ordered networks and a parallel was evidenced between these structures and molecular assemblies described in liquid crystals. Similar structures are now obtained in vitro. Indeed, when purified type I collagen is highly concentrated in an acid soluble state, the protein spontaneously assembles into ordered liquid crystalline phases. After a sol/gel transition triggered by pH increase, biomimetic materials are formed which resemble the exact compact bone matrix architecture over distances reaching centimetres and more. The properties of these highly ordered materials will be reviewed recalling their supramolecular arrangement and the corresponding patterns when visualised in polarised light microscopy (birefringence) and transmission electron microscopy (TEM). The association of inorganic phases (amorphous silica) to form chiral hybrid materials will also be described so as the behaviour of cells (fibroblast adhesion and migration) when seeded on these dense biomimetic matrices.

Flagellar ejection is tightly coupled to the cell cycle in Caulobacter crescentus. The MS ring protein FliF, which anchors the flagellar structure in the inner membrane, is degraded coincident with flagellar release. Previous work showed that removal of 26 amino acids from the C terminus of FliF prevents degradation of the protein and interferes with flagellar assembly. To understand FliF degradation in more detail, we identified the protease responsible for FliF degradation and performed a high-resolution mutational analysis of the C-terminal degradation signal of FliF. Cell cycle-dependent turnover of FliF requires an intact clpA gene, suggesting that the ClpAP protease is required for removal of the MS ring protein. Deletion analysis of the entire C-terminal cytoplasmic portion of FliF C confirmed that the degradation signal was contained in the last 26 amino acids that were identified previously. However, only deletions longer than 20 amino acids led to a stable FliF protein, while shorter deletions dispersed over the entire 26 amino acids critical for turnover had little effect on stability. This indicated that the nature of the degradation signal is not based on a distinct primary amino acid sequence. The addition of charged amino acids to the C-terminal end abolished cell cycle-dependent FliF degradation, implying that a hydrophobic tail feature is important for the degradation of FliF. Consistent with this, ClpA-dependent degradation was restored when a short stretch of hydrophobic amino acids was added to the C terminus of stable FliF mutant forms.

The in vitro apatite-forming ability of poly(epsilon-caprolactone) (70 wt %)-silica sol-gels, with hydroxyl and triethoxysilane end-groups with potential use in bone repair have been assessed using static and dynamic osteoconductivity tests. Diffuse reflectance infrared spectroscopy, scanning electron microscopy, and microanalysis techniques were used to observe and characterize precipitates formed on the material's surface. An apatite layer was observed to form on both of the composites' surface. However, variations were observed according to the test method used, in accordance with other studies on bioactive ceramics. A third method, the "alternate soaking process" (ASP) was developed to provide rapid results and quantify the amount of calcium-containing precipitates formed on the surface of potentially "bioactive" materials. The results presented here show that for a material to be bioactive and have the ability to form a precipitate containing calcium and phosphate ions, levels of calcium ions measured by a complexometric assay should be significantly higher than the level of 5 microg/cm(2). This level of calcium ions was obtained after 20 ASP cycles for the hydroxyl and triethoxysilane end-capped poly(epsilon-caprolactone) samples that did not form precipitates on their surfaces even after >50 ASP cycles. For the two sol-gel silica composites containing approximately 70% hydroxyl and triethoxysilane poly(epsilon-caprolactone), there was no significant difference in the amount of calcium-containing precipitate as observed using the in vitro apatite-forming ability tests suggesting that polymer end-group modification is not detrimental to the apatite-forming ability of such composites., (Copyright 2004 Wiley Periodicals, Inc.)