Prophylaxis of implant-related infections by local release of vancomycin from a hydrogel in rabbits.

Local prophylaxis with antibiotic-loaded bone cement is a successful method to prevent post-operative infections in patients receiving orthopaedic implants. No comparable method is available for uncemented implants. Therefore, a hydrogel consisting of hyaluronic and polylactic acids was evaluated in a rabbit model for delivery of antimicrobial agents to prevent post-operative infections. In a pilot study, the suitability of the in vivo model was assessed by testing the hydrogel as carrier material for antimicrobial agents.In the main study, the antimicrobial-agent-loaded hydrogel was evaluated for infection prophylaxis. Rabbits received a titanium rod intramedullary in the tibia after contamination with Staphylococcus aureus. The rods were coated with unloaded hydrogel (Gel), hydrogel loaded with 2 % (Van2) or 5 % vancomycin (Van5), bioactive glass (BAG) or N-acetyl-L-cysteine (NAC). To analyse the infection severity after 28 d, histopathological, bacteriological, micro-computed tomographic and haematological analyses were performed. In the pilot study, the Van5 group had less infection (0/6 infected) as compared to the Gel group (5/5, p = 0.000) and the in vivo model was deemed suitable. In the main study, in the Van2 and Van5 groups, the number of infected animals was lower [1/6 (p = 0.006) and 2/6 (p = 0.044) infected, respectively]. In contrast, BAG and NAC groups showed no infection reduction (5/6 both groups, p = 0.997). The hydrogel can be used as a local carrier of vancomycin for prophylaxis of implant-related infections.The present study showed promising results for local delivery of antibacterial agents by hydrogel to prevent implant-related infections.

The chondrogenic differentiation potential of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are particularly promising for tissue engineering (TE) due to the ease of their isolation procedure, great expansion potential and capability to differentiate towards several cell types of the mesodermal, ectodermal and endodermal lineages. Although several studies hint that DPSCs exhibit potential for cartilage tissue formation, the chondrogenic potential of DPSCs has only been marginally explored. Thus, the aim of the present study was to closely investigate the chondrogenic differentiation capacity of DPSCs for TE applications. More specifically, the potential of DPSCs for engineering hyaline and fibrous cartilage was determined. DPSCs obtained from 7 human molars were expanded and chondrogenically differentiated in a 3D pellet culture model. After 21 d of differentiation with chondrogenic stimuli, DPSCs displayed glycosaminoglycan, aggrecan and limited collagen type II deposition. Cells presented an elongated morphology and produced a collagen-rich extracellular matrix, with a predominance of collagen type I in most of the samples, a characteristic of fibrous cartilage tissue. Variations in the administration periods of several chondro-inductive growth factors, including transforming growth factor beta 3, bone morphogenetic protein-2, -6, -7 and insulin-like growth factor-1, did not increase glycosaminoglycan or collagen type II deposition, typical markers of hyaline cartilage tissue. Furthermore, DPSCs could not be stimulated to go into hypertrophic chondrogenesis. These results indicated that under a large variety of chondro-inductive culture conditions, DPSCs could form fibrocartilaginous tissues but not hyaline cartilage. Thus, DPSCs represent a valuable cell source for the regeneration of fibrocartilage in joints.

Complete regeneration of large bone defects in rats with commercially available fibrin loaded with BMP-2.

This study aimed at investigating in vitro and in vivo the efficiency of commercially available fibrin as a carrier for controlled and sustained bone morphogenetic protein-2 (BMP-2) release to induce bone formation and reduce the side effects of its use. In vitro release and activity of low-dose recombinant human BMP-2 (rhBMP-2) (37.5 µg/mL) embedded in commercially available fibrin were evaluated and, subsequently, critical-size femur defects in rats were grafted to study bone regeneration and vascularisation by micro-computed tomography (µCT) and histology. In vitro experiments showed a sustained BMP-2 release with a high BMP activity remaining after 28 d. In vivo, fibrin loaded with BMP-2 showed an extremely fast bone healing, with a large amount of new bone formation throughout the entire defect in the first 4 weeks and complete cortical repair and fusion after 8 weeks, with no ectopic bone formation. In contrast, the control fibrin group did not fuse after 12 weeks. Vascularisation was similar in both groups at 4 and 12 weeks after implantation. In conclusion, commercially available fibrin is a very efficient carrier for rhBMP-2 to graft critical-size cortical bone defects and might be a more optimal delivery vehicle for BMP-2-induced bone regeneration than currently available collagen sponges.

Engineering of a complex bone tissue model with endothelialised channels and capillary-like networks.

In engineering of tissue analogues, upscaling to clinically-relevant sized constructs remains a significant challenge. The successful integration of a vascular network throughout the engineered tissue is anticipated to overcome the lack of nutrient and oxygen supply to residing cells. This work aimed at developing a multiscale bone-tissue-specific vascularisation strategy. Engineering pre-vascularised bone leads to biological and fabrication dilemmas. To fabricate channels endowed with an endothelium and suitable for osteogenesis, rather stiff materials are preferable, while capillarisation requires soft matrices. To overcome this challenge, gelatine-methacryloyl hydrogels were tailored by changing the degree of functionalisation to allow for cell spreading within the hydrogel, while still enabling endothelialisation on the hydrogel surface. An additional challenge was the combination of the multiple required cell-types within one biomaterial, sharing the same culture medium. Consequently, a new medium composition was investigated that simultaneously allowed for endothelialisation, capillarisation and osteogenesis. Integrated multipotent mesenchymal stromal cells, which give rise to pericyte-like and osteogenic cells, and endothelial-colony-forming cells (ECFCs) which form capillaries and endothelium, were used. Based on the aforementioned optimisation, a construct of 8 × 8 × 3 mm, with a central channel of 600 µm in diameter, was engineered. In this construct, ECFCs covered the channel with endothelium and osteogenic cells resided in the hydrogel, adjacent to self-assembled capillary-like networks. This study showed the promise of engineering complex tissue constructs by means of human primary cells, paving the way for scaling-up and finally overcoming the challenge of engineering vascularised tissues.

Hyaluronic Acid-Based Hydrogel Coating Does Not Affect Bone Apposition at the Implant Surface in a Rabbit Model.

BACKGROUND: Uncemented orthopaedic implants rely on the bone-implant interface to provide stability, therefore it is essential that a coating does not interfere with the bone-forming processes occurring at the implant interface. In addition, local application of high concentrations of antibiotics for prophylaxis or treatment of infection may be toxic for osteoblasts and could impair bone growth. QUESTIONS/PURPOSES: In this animal study, we investigated the effect of a commercially available hydrogel, either unloaded or loaded with 2% vancomycin. We asked, does unloaded hydrogel or hydrogel with vancomycin (1) interfere with bone apposition and timing of bone deposition near the implant surface; and (2) induce a local or systemic inflammatory reaction as determined by inflammation around the implant and hematologic parameters. METHODS: In 18 New Zealand White rabbits, an uncoated titanium rod (n = 6), a rod coated with unloaded hydrogel (n = 6), or a rod coated with 2% vancomycin-loaded hydrogel (n = 6) was implanted in the intramedullary canal of the left tibia. After 28 days, the bone volume fraction near the implant was measured with microCT analysis, inflammation was semiquantitatively scored on histologic sections, and timing of bone apposition was followed by semiquantitative scoring of fluorochrome incorporation on histologic sections. Two observers, blinded to the treatment, scored the sections and reconciled their scores if there was a disagreement. The hematologic inflammatory reaction was analyzed by measuring total and differential leukocyte counts and erythrocyte sedimentation rates in blood. With group sizes of six animals per group, we had 79% power to detect a difference of 25% in histologic scoring for infection and inflammation. RESULTS: No differences were found in the amount of bone apposition near the implant in the No Gel group (48.65% ± 14.95%) compared with the Gel group (59.97% ± 5.02%; mean difference [MD], 11.32%; 95% CI, -3.89% to 26.53%; p = 0.16) or for the Van2 group (56.12% ± 10.06%; MD, 7.46; 95% CI, -7.75 to 22.67; p = 0.40), with the numbers available. In addition, the scores for timing of bone apposition did not differ between the No Gel group (0.50 ± 0.55) compared with the Gel group (0.33 ± 0.52; MD, -0.17; 95% CI, -0.86 to 0.53; p = 0.78) or the Van2 group (0.83 ± 0.41; MD, 0.33; 95% CI, -0.36 to 1.03; p = 0.42). Furthermore, we detected no differences in the histopathology scores for inflammation in the No Gel group (2.33 ± 1.67) compared with the Gel group (3.17 ± 1.59; MD, 0.83; 95% CI, -0.59 to 2.26; p = 0.31) or to the Van2 group (2.5 ± 1.24; MD, 0.17; 95% CI, -1.26 to 1.59; p = 0.95). Moreover, no differences in total leukocyte count, erythrocyte sedimentation rate, and neutrophil, monocyte, eosinophil, basophil, and lymphocyte counts were present between the No Gel or Van2 groups compared with the Gel control group, with the numbers available. CONCLUSION: The hydrogel coated on titanium implants, unloaded or loaded with 2% vancomycin, had no effect on the volume or timing of bone apposition near the implant, and did not induce an inflammatory reaction in vivo, with the numbers available. CLINICAL RELEVANCE: Antibiotic-loaded hydrogel may prove to be a valuable option to protect orthopaedic implants from bacterial colonization. Future clinical safety studies will need to provide more evidence that this product does not impair bone formation near the implant and prove the safety of this product.

Trained innate immunity modulates osteoblast and osteoclast differentiation.

Macrophages are key regulators in bone repair and regeneration. Recent studies have shown that long-term epigenetic changes and metabolic shifts occur during specific immune training of macrophages that affect their functional state, resulting in heightened (trained) or reduced (tolerant) responses upon exposure to a second stimulus. This is known as innate immune memory. Here, we study the impact of macrophages' memory trait on osteoblast differentiation of human mesenchymal stromal cells (hMSCs) and osteoclast differentiation. An in vitro trained immunity protocol of monocyte-derived macrophages was employed using inactivated Candida albicans and Bacillus Calmette-Guérin (BCG) to induce a 'trained' state and Pam3CSK4 (PAM) and Lipopolysaccharides (LPS) to induce a 'tolerance' state. Macrophages were subsequently cocultured with hMSCs undergoing osteogenic differentiation during either resting (unstimulated) or inflammatory conditions (restimulated with LPS). Alkaline phosphatase activity, mineralization, and cytokine levels (TNF, IL-6, oncostatin M and SDF-1α) were measured. In addition, macrophages underwent osteoclast differentiation. Our findings show that trained and tolerized macrophages induced opposing results. Under resting conditions, BCG-trained macrophages enhanced ALP levels (threefold), while under inflammatory conditions this was found in the LPS-tolerized macrophages (fourfold). Coculture of hMSCs with trained macrophages showed mineralization while tolerized macrophages inhibited the process under both resting and inflammatory conditions. While osteoclast differentiation was not affected in trained-macrophages, this ability was significantly loss in tolerized ones. This study further confirms the intricate cross talk between immune cells and bone cells, highlighting the need to consider this interaction in the development of personalized approaches for bone regenerative medicine.

Intact vitreous humor as a potential extracellular matrix hydrogel for cartilage tissue engineering applications.

Decellularisation of tissues, utilising their biochemical cues, poses exciting tissue engineering (TE) opportunities. However, removing DNA from cartilage (dCart) requires harsh treatments due to its dense structure, causing loss of bioactivity and limiting its application as a cartilaginous extra cellular matrix (ECM). In this study, we demonstrate for the first time the successful application of vitreous humor (VH), a highly hydrated tissue closely resembling the glycosaminoglycan (GAG) and collagen composition of cartilage, as an ECM hydrogel to support chondrogenic differentiation. Equine VH was extracted followed by biochemical quantifications, histological examinations, cytotoxicity (human mesenchymal stromal cells, hMSCs and human articular chondrocytes, hACs) and U937 cell proliferation studies. VH was further seeded with hACs or hMSCs and cultured for 3-weeks to study chondrogenesis compared to scaffold-free micro-tissue pellet cultures and collagen-I hydrogels. Viability, metabolic activity, GAG and DNA content, chondrogenic gene expression (aggrecan, collagen I/II mRNA) and mechanical properties were quantified and matrix deposition was visualised using immunohistochemistry (Safranin-O, collagen I/II). VH was successfully extracted, exhibiting negligible amounts of DNA (0.4 ±â€¯0.4 µg/mg dry-weight) and notable preservation of ECM components. VH displayed neither cytotoxic responses nor proliferation of macrophage-like U937 cells, instead enhancing both hMSC and hAC proliferation. Interestingly, encapsulated cells self-assembled the VH-hydrogel into spheroids, resulting in uniform distribution of both GAGs and collagen type II with increased compressive mechanical properties, rendering VH a permissive native ECM source to fabricate cartilaginous hydrogels for potential TE applications. STATEMENT OF SIGNIFICANCE: Fabricating bioactive and cell-instructive cartilage extracellular matrix (ECM) derived biomaterials and hydrogels has over recent years proven to be a challenging task, often limited by poor retention of inherent environmental cues post decellularisation due to the dense and avascular nature of native cartilage. In this study, we present an alternative route to fabricate highly permissive and bioactive ECM hydrogels from vitreous humor (VH) tissue. This paper specifically reports the discovery of optimal VH extraction protocols and cell seeding strategy enabling fabrication of cartilaginous matrix components into a hydrogel support material for promoting chondrogenic differentiation. The work showcases a naturally intact and unmodified hydrogel design that improves cellular responses and may help guide the development of cell instructive and stimuli responsive hybrid biomaterials in a number of TERM applications.

The impact of immune response on endochondral bone regeneration.

Tissue engineered cartilage substitutes, which induce the process of endochondral ossification, represent a regenerative strategy for bone defect healing. Such constructs typically consist of multipotent mesenchymal stromal cells (MSCs) forming a cartilage template in vitro, which can be implanted to stimulate bone formation in vivo. The use of MSCs of allogeneic origin could potentially improve the clinical utility of the tissue engineered cartilage constructs in three ways. First, ready-to-use construct availability can speed up the treatment process. Second, MSCs derived and expanded from a single donor could be applied to treat several patients and thus the costs of the medical interventions would decrease. Finally, it would allow more control over the quality of the MSC chondrogenic differentiation. However, even though the envisaged clinical use of allogeneic cell sources for bone regeneration is advantageous, their immunogenicity poses a significant obstacle to their clinical application. The aim of this review is to increase the awareness of the role played by immune cells during endochondral ossification, and in particular during regenerative strategies when the immune response is altered by the presence of implanted biomaterials and/or cells. More specifically, we focus on how this balance between immune response and bone regeneration is affected by the implantation of a cartilaginous tissue engineered construct of allogeneic origin.

Development of a thermosensitive HAMA-containing bio-ink for the fabrication of composite cartilage repair constructs.

Fine-tuning of bio-ink composition and material processing parameters is crucial for the development of biomechanically relevant cartilage constructs. This study aims to design and develop cartilage constructs with tunable internal architectures and relevant mechanical properties. More specifically, the potential of methacrylated hyaluronic acid (HAMA) added to thermosensitive hydrogels composed of methacrylated poly[N-(2-hydroxypropyl)methacrylamide mono/dilactate] (pHPMA-lac)/polyethylene glycol (PEG) triblock copolymers, to optimize cartilage-like tissue formation by embedded chondrocytes, and enhance printability was explored. Additionally, co-printing with polycaprolactone (PCL) was performed for mechanical reinforcement. Chondrocyte-laden hydrogels composed of pHPMA-lac-PEG and different concentrations of HAMA (0%-1% w/w) were cultured for 28 d in vitro and subsequently evaluated for the presence of cartilage-like matrix. Young's moduli were determined for hydrogels with the different HAMA concentrations. Additionally, hydrogel/PCL constructs with different internal architectures were co-printed and analyzed for their mechanical properties. The results of this study demonstrated a dose-dependent effect of HAMA concentration on cartilage matrix synthesis by chondrocytes. Glycosaminoglycan (GAG) and collagen type II content increased with intermediate HAMA concentrations (0.25%-0.5%) compared to HAMA-free controls, while a relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. Young's moduli of generated hydrogel constructs ranged from 14 to 31 kPa and increased with increasing HAMA concentration. The pHPMA-lac-PEG hydrogels with 0.5% HAMA were found to be optimal for cartilage-like tissue formation. Therefore, this hydrogel system was co-printed with PCL to generate porous or solid constructs with different mesh sizes. Young's moduli of these composite constructs were in the range of native cartilage (3.5-4.6 MPa). Interestingly, the co-printing procedure influenced the mechanical properties of the final constructs. These findings are relevant for future bio-ink development, as they demonstrate the importance of selecting proper HAMA concentrations, as well as appropriate print settings and construct designs for optimal cartilage matrix deposition and final mechanical properties of constructs, respectively.

Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics.

Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone.

Selection of an optimal antiseptic solution for intraoperative irrigation: an in vitro study.

BACKGROUND: With increasing bacterial antibiotic resistance and an increased infection risk due to more complicated surgical procedures and patient populations, prevention of surgical infection is of paramount importance. Intraoperative irrigation with an antiseptic solution could provide an effective way to reduce postoperative infection rates. Although numerous studies have been conducted on the bactericidal or cytotoxic characteristics of antiseptics, the combination of these characteristics for intraoperative application has not been addressed. METHODS: Bacteria (Staphylococcus aureus and S. epidermidis) and human cells were exposed to polyhexanide, hydrogen peroxide, octenidine dihydrochloride, povidone-iodine, and chlorhexidine digluconate at various dilutions for two minutes. Bactericidal properties were calculated by means of the quantitative suspension method. The cytotoxic effect on human fibroblasts and mesenchymal stromal cells was determined by a WST-1 metabolic activity assay. RESULTS: All of the antiseptics except for polyhexanide were bactericidal and cytotoxic at the commercially available concentrations. When diluted, only povidone-iodine was bactericidal at a concentration at which some cell viability remained. The other antiseptics tested showed no cellular survival at the minimal bactericidal concentration. CONCLUSIONS: Povidone-iodine diluted to a concentration of 1.3 g/L could be the optimal antiseptic for intraoperative irrigation. This should be established by future clinical studies.

Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.

OBJECTIVE: Although extracellular matrix (ECM)-derived scaffolds have been extensively studied and applied in a number of clinical applications, the use of ECM as a biomaterial for (osteo)chondral regeneration is less extensively explored. This study aimed at evaluating the chondrogenic potential of cells seeded on cartilage-derived matrix (CDM) scaffolds in vitro. DESIGN: Scaffolds were generated from decellularized equine articular cartilage and seeded with either chondrocytes or multipotent stromal cells (MSCs). After 2, 4, and 6 weeks of in vitro culture, CDM constructs were analyzed both histologically (hematoxylin and eosin, Safranin-O, collagen types I and II) and biochemically (glycosaminoglycan [GAG] and DNA content). RESULTS: After 4 weeks, both cell types demonstrated chondrogenic differentiation; however, the MSCs significantly outperformed chondrocytes in producing new GAG-containing cartilaginous matrix. CONCLUSION: These promising in vitro results underscore the potency of CDM scaffolds in (osteo)chondral defect repair.

In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells.

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing "bulk-cultured" BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

Numerical analysis of ischemia- and compression-induced injury in tissue-engineered skeletal muscle constructs.

Pressure-related deep tissue injury may develop in skeletal muscle tissue which is subjected to prolonged compression. For early detection, it is important to understand the underlying damage processes. Gawlitta et al. [Gawlitta, D., C. W. J. Oomens, D. L. Bader, F. P. T. Baaijens, and C. V. C. Bouten. Temporal differences in the influence of ischemic factors and deformation on the metabolism of engineered skeletal muscle. J. Appl. Physiol. 103(2):464-473, 2007b] subjected tissue-engineered muscle constructs to ischemia and deformation to study their effects on viability. Contrary to previous findings, no decrease in viability was found due to compression. However, the nature of their measurement method complicated interpretation of the results, particulary when deformation was involved. Changes in the constructs were assessed by measurements in the surrounding medium. The theoretical model developed in the present study describes metabolism, diffusion, and cell death in the experiments, and accounts for reduced diffusion due to compression. It was demonstrated that the lack of effect of compression on tissue viability, as measured in the experiments, could be explained by the compression-induced decrease in diffusivity. Compression did lead to considerable cell death but this could not be measured by Gawlitta et al. [Gawlitta, D., C. W. J. Oomens, D. L. Bader, F. P. T. Baaijens, and C. V. C. Bouten. Temporal differences in the influence of ischemic factors and deformation on the metabolism of engineered skeletal muscle. J. Appl. Physiol. 103(2):464-473, 2007b] because diffusion of the cell death marker to the medium was limited. This study shows that a proper description of transport processes is essential for a correct interpretation of experiments in which indirect measurement methods are used.