Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells.

PURPOSE: This study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization. METHODS: hAFCs from spinal trauma patients were stimulated with TNF-α or IL-1ß. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 µM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model. RESULTS: IL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1ß treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation. CONCLUSION: Cxb can inhibit PGE-2 production in hAFCs in an IL-1ß-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

Artificial Intelligence Accurately Detects Traumatic Thoracolumbar Fractures on Sagittal Radiographs.

Background and Objectives: Commonly being the first step in trauma routine imaging, up to 67% fractures are missed on plain radiographs of the thoracolumbar (TL) spine. The aim of this study was to develop a deep learning model that detects traumatic fractures on sagittal radiographs of the TL spine. Identifying vertebral fractures in simple radiographic projections would have a significant clinical and financial impact, especially for low- and middle-income countries where computed tomography (CT) and magnetic resonance imaging (MRI) are not readily available and could help select patients that need second level imaging, thus improving the cost-effectiveness. Materials and Methods: Imaging studies (radiographs, CT, and/or MRI) of 151 patients were used. An expert group of three spinal surgeons reviewed all available images to confirm presence and type of fractures. In total, 630 single vertebra images were extracted from the sagittal radiographs of the 151 patients-302 exhibiting a vertebral body fracture, and 328 exhibiting no fracture. Following augmentation, these single vertebra images were used to train, validate, and comparatively test two deep learning convolutional neural network models, namely ResNet18 and VGG16. A heatmap analysis was then conducted to better understand the predictions of each model. Results: ResNet18 demonstrated a better performance, achieving higher sensitivity (91%), specificity (89%), and accuracy (88%) compared to VGG16 (90%, 83%, 86%). In 81% of the cases, the "warm zone" in the heatmaps correlated with the findings, suggestive of fracture within the vertebral body seen in the imaging studies. Vertebras T12 to L2 were the most frequently involved, accounting for 48% of the fractures. A4, A3, and A1 were the most frequent fracture types according to the AO Spine Classification. Conclusions: ResNet18 could accurately identify the traumatic vertebral fractures on the TL sagittal radiographs. In most cases, the model based its prediction on the same areas that human expert classifiers used to determine the presence of a fracture.

Serum biomarkers for Modic changes in patients with chronic low back pain.

PURPOSE: Lumbar Modic change (MC) can serve as a diagnostic marker as well as an independent source of chronic low back pain (CLBP). This study aimed to test for the existence of serum biomarkers in CLBP patients with MC. METHODS: Age- and sex-matched CLBP patients with confirmed MC on lumbar MRI (n = 40) and pain-free controls (n = 40) were assessed. MC was classified into M1, predominating M1, predominating M2 and M2. MC volumes were calculated. Fasting blood samples were assessed for inflammatory mediators, signalling molecules, growth factors and bone turnover markers. Serum concentrations of 46 biomarkers were measured. RESULTS: Median concentrations of interleukin (IL)-15 (p < 0.001), IL-8 (p < 0.001), tumour necrosis factor (TNF)-alpha (p < 0.001), Eotaxin-1 (p < 0.05), Eotaxin-3 (p < 0.001), monocyte chemotactic protein (MCP)-1 (p < 0.05), macrophage inflammatory protein (MIP)-1alpha (p < 0.01), TEK receptor tyrosine kinase (Tie)-2 (p < 0.001), vascular cell adhesion molecule (VCAM)-1 (p < 0.001), RANTES (p < 0.001), C telopeptide of type I collagen (CTX)-1 (p < 0.001), vascular endothelial growth factor (VEGF)-C (p < 0.001), VEGF-D (p < 0.05), fms-related tyrosine kinase (Flt)-1 (p < 0.01) and intercellular adhesion molecule (ICAM)-1 (p < 0.01) were significantly higher among controls. IL-1sRII (23.2 vs. 15.5 ng/ml, p < 0.001) and hepatocyte growth factor (HGF)-1 (169 vs. 105 pg/ml, p < 0.01) concentrations were significantly higher among patients. Type or volume of MC was not associated with biomarker concentrations. CONCLUSIONS: This is the first study to assess the blood serum biomarker profile in individuals with CLBP with MC. Several biomarkers were suppressed, while two markers (IL-1sRII and HGF) were elevated among MC patients, irrespective of MC type or size, with CLBP compared with asymptomatic controls.

A Hyaluronan and Platelet-Rich Plasma Hydrogel for Mesenchymal Stem Cell Delivery in the Intervertebral Disc: An Organ Culture Study.

The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 µL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~70%) compared to the hydrogel without hMSCs. Furthermore, disc morphology was maintained, and the hydrogel showed signs of integration with the surrounding tissues. At the gene expression level, the hydrogel loaded with hMSCs preserved the normal metabolism of the tissue. The IVD environment promoted hMSC differentiation towards a NP cell phenotype by increasing cytokeratin-19 (KRT19) gene expression. This study demonstrated that the hydrogel composed of HA/PRP/BTX represents a valid carrier for hMSCs being able to maintain a good cell viability while stimulating cell activity and NP marker expression.

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1ß, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

Dexamethasone Induces Changes in Osteogenic Differentiation of Human Mesenchymal Stromal Cells via SOX9 and PPARG, but Not RUNX2.

Despite the huge body of research on osteogenic differentiation and bone tissue engineering, the translation potential of in vitro results still does not match the effort employed. One reason might be that the protocols used for in vitro research have inherent pitfalls. The synthetic glucocorticoid dexamethasone is commonly used in protocols for trilineage differentiation of human bone marrow mesenchymal stromal cells (hBMSCs). However, in the case of osteogenic commitment, dexamethasone has the main pitfall of inhibiting terminal osteoblast differentiation, and its pro-adipogenic effect is well known. In this work, we aimed to clarify the role of dexamethasone in the osteogenesis of hBMSCs, with a particular focus on off-target differentiation. The results showed that dexamethasone does induce osteogenic differentiation by inhibiting SOX9 expression, but not directly through RUNX2 upregulation as it is commonly thought. Rather, PPARG is concomitantly and strongly upregulated, leading to the formation of adipocyte-like cells within osteogenic cultures. Limiting the exposure to dexamethasone to the first week of differentiation did not affect the mineralization potential. Gene expression levels of RUNX2, SOX9, and PPARG were simulated using approximate Bayesian computation based on a simplified theoretical model, which was able to reproduce the observed experimental trends but with a different range of responses, indicating that other factors should be integrated to fully understand how dexamethasone influences cell fate. In summary, this work provides evidence that current in vitro differentiation protocols based on dexamethasone do not represent a good model, and further research is warranted in this field.

Intervertebral disc organ culture for the investigation of disc pathology and regeneration - benefits, limitations, and future directions of bioreactors.

Low back pain is the leading cause of disability worldwide and in many patients the source of pain can be attributed to pathological changes within the intervertebral disc (IVD). As present treatment options fail to address the underlying biological problem, novel therapies are currently subject to intense research. The physiologic IVD microenvironment features a highly complex interaction of biochemical and mechanical factors influencing cell metabolism and extracellular matrix turnover and is therefore difficult to simulate for research purposes on IVD pathology. The first whole organ culture models were not able to sufficiently replicate human in vivo conditions as mechanical loading, the predominant way of IVD nutrient supply and waste exchange, remained disregarded. To mimic the unique IVD niche more realistically, whole organ culture bioreactors have been developed, allowing for dynamic loading of IVDs and nutrient exchange. Recent advancements on bioreactor systems have facilitated whole organ culture of various IVDs for extended periods. IVD organ culture bioreactors have the potential to bridge the gap between in vitro and in vivo systems and thus may give valuable insights on IVD pathology and/or potential novel treatment approaches if the respective model is adjusted according to a well-defined research question. In this review, we outline the potential of currently utilized IVD bioreactor systems and present suggestions for further developments to more reliably investigate IVD biology and novel treatment approaches.

Regulation of Inflammatory Response in Human Osteoarthritic Chondrocytes by Novel Herbal Small Molecules.

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1ß/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2''-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects.

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG-HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14-91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45-CD34-CD73+, CD45-CD34-CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment.

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.

Cross-Linking Chemistry of Tyramine-Modified Hyaluronan Hydrogels Alters Mesenchymal Stem Cell Early Attachment and Behavior.

Given the significance of hydrogels as cell-instructive materials, it is important to understand how differences in their chemical and physical properties are able to direct cell fate. For example, it remains unclear how different hydrogel cross-linking chemistries and gelation mechanisms influence cell behavior. Here, we report on hyaluronan-tyramine (HA-Tyr) hydrogels prepared either with enzymatic cross-linking using horseradish peroxidase and H2O2 or with visible light (500 nm) triggered gelation. We demonstrate that when hydrogels are polymerized to equivalent Young's moduli, the specific cross-linking chemistry of HA-Tyr hydrogels can have a substantial impact on mesenchymal stem cell (MSC) behavior. MSCs cultured on HA-Tyr hydrogels exhibit increased cell spread areas on enzymatically formed substrates relative to photo-cross-linked matrices. While enzymatically formed hydrogels led to MSCs exhibiting greater cell focal adhesion length, MSCs cultured on the photo-cross-linked matrices exhibited smaller cell spread area and shorter focal adhesion length but generated increased traction stress. These findings highlight the importance of understanding hydrogel cross-linking chemistries when the role of biophysical cues in regulating stem cell fate is investigated.

Self-Healing Dynamic Hydrogel as Injectable Shock-Absorbing Artificial Nucleus Pulposus.

The intervertebral discs (IVDs) provide unique flexibility to the spine and exceptional shock absorbing properties under impact. The inner core of the IVD, the nucleus pulposus (NP) is responsible for this adaptive behavior. Herein, we evaluate an injectable, self-healing dynamic hydrogel (DH) based on gold(I)-thiolate/disulfide (Au-S/SS) exchange as NP replacement in a spine motion segment model. For the first time, we report the application of dynamic covalent hydrogels inside biological tissues. The dynamic exchange between Au-S species and disulfide bonds (SS) resulted in self-healing ability and frequency-dependent stiffness of the hydrogel, which was also confirmed in spine motion segments. Injection of preformed DH into nucleotomized IVDs restored the full biomechanical properties of intact IVDs, including the stiffening effect observed at increasing frequencies, which cannot be achieved with conventional covalent hydrogel. DH has the potential to counteract IVD degeneration associated with high frequency vibrations. Self-healing properties, confirmed by rheology studies and macroscopic observation after injection, were required to inject preformed DH, which recovered its mechanical integrity and microstructure to act as an artificial NP. On the other hand, covalent hydrogel did not show any restoration of NP properties as this conventional material suffered irreversible damages after injection, which demonstrates that the dynamic properties are crucial for this application. The persistence of DH in the IVD space following cyclic high-frequency loading, confirmed by tomography after mechanical testing, suggests that this material would have long life span as an injectable NP replacement material.

Platelet-Rich Plasma as an Autologous and Proangiogenic Cell Delivery System.

Angiogenesis is a key factor in early stages of wound healing and is crucial for the repair of vascularized tissues such as the bone. However, supporting timely revascularization of the defect site still presents a clinical challenge. Tissue engineering approaches delivering endothelial cells or prevascularized constructs may overcome this problem. In the current study, we investigated platelet-rich plasma (PRP) gels as autologous, injectable cell delivery systems for prevascularized constructs. PRP was produced from human thrombocyte concentrates. GFP-expressing human umbilical vein endothelial cells (HUVECs) and human bone marrow-derived mesenchymal stem cells (MSCs) were encapsulated in PRP gels in different proportions. The formation of cellular networks was assessed over 14 days by time-lapse microscopy, gene expression analysis, and immunohistology. PRP gels presented a favorable environment for the formation of a three-dimensional (3D) cellular network. The formation of these networks was apparent as early as 3 days after seeding. Networks increased in complexity and branching over time but were only stable in HUVEC-MSC cocultures. The high cell viability together with the 3D capillary-like networks observed at early time points suggests that PRP can be used as an autologous and proangiogenic cell delivery system for the repair of vascularized tissues such as the bone.

Development of an ex vivo cavity model to study repair strategies in loaded intervertebral discs.

PURPOSE: The aim of this study was to compare two approaches for the delivery of biomaterials to partially nucleotomised intervertebral discs in whole organ culture under loading. Such models can help to bridge the gap between in vitro and in vivo studies by assessing (1) suitability of biomaterial delivery and defect closure methods, (2) effect of mechanical loading and (3) tissue response. METHODS: Mechanical performance of bovine discs filled with a hyaluronan-based thermoreversible hydrogel delivered through the annulus fibrosus (AF) or the bony endplate (EP) was evaluated under cyclic axial loading in a bioreactor. The loading protocol was optimised to achieve physiological disc height changes in nucleotomised discs. A loading regime of 0.06 ± 0.02 MPa, 0.1 Hz, 6 h daily was applied on the nucleotomised discs. Disc height and stiffness were tracked for 5 days, followed by histological analyses. RESULTS: Creation of a defect is less demanding for AF approach, while sealing is superior with the EP approach. Dynamic compressive stiffness is reduced following nucleotomy, with no significant difference between the two approaches. Disc height loss was higher, disc height recovery was lower and region around the defect with reduced cell viability was smaller for AF-approached than EP-approached discs. CONCLUSIONS: Two alternative methods for biomaterial testing in whole organ culture under loading were developed. Such models bring insights on the ability of the biomaterial to restore the mechanical behaviour of the discs. From a clinical perspective, the cavity models can simulate treatment of nucleotomy after disc herniation in young patients, whereby the remaining nucleus pulposus is still functional and therefore at high risk of re-herniation, though the defect may differ from the clinical situation.

Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic.

Mesenchymal stem cells (MSCs) are increasingly being used in tissue engineering and cell-based therapies in all fields ranging from orthopedic to cardiovascular medicine. Despite years of research and numerous clinical trials, MSC therapies are still very much in development and not considered mainstream treatments. The majority of approaches rely on an in vitro cell expansion phase in monolayer to produce large cell numbers prior to implantation. It is clear from the literature that this in vitro expansion phase causes dramatic changes in MSC phenotype which has very significant implications for the development of effective therapies. Previous reviews have sought to better characterize these cells in their native and in vitro environments, described known stem cell interactions within the bone marrow, and discussed the use of innovative culture systems aiming to model the bone marrow stem cell niche. The purpose of this review is to provide an update on our knowledge of MSCs in their native environment, focusing on bone marrow-derived MSCs. We provide a detailed description of the differences between naive cells and those that have been cultured in vitro and examine the effect of isolation and culture parameters on these phenotypic changes. We explore the concept of "one step" MSC therapy and discuss the potential cellular and clinical benefits. Finally, we describe recent work attempting to model the MSC bone marrow niche, with focus on both basic research and clinical applications and consider the challenges associated with these new generation culture systems.

Three-dimensional culture and characterization of mononuclear cells from human bone marrow.

BACKGROUND AIMS: The diverse phenotypic changes and clinical and economic disadvantages associated with the monolayer expansion of bone marrow-derived mesenchymal stromal cells (MSCs) have focused attention on the development of one-step intraoperative cells therapies and homing strategies. The mononuclear cell fraction of bone marrow, inclusive of discrete stem cell populations, is not well characterized, and we currently lack suitable cell culture systems in which to culture and investigate the behavior of these cells. METHODS: Human bone marrow-derived mononuclear cells were cultured within fibrin for 2 weeks with or without fibroblast growth factor-2 supplementation. DNA content and cell viability of enzymatically retrieved cells were determined at days 7 and 14. Cell surface marker profiling and cell cycle analysis were performed by means of multi-color flow cytometry and a 5-ethynyl-2'-deoxyuridine incorporation assay, respectively. RESULTS: Total mononuclear cell fractions, isolated from whole human bone marrow, was successfully cultured in fibrin gels for up to 14 days under static conditions. Discrete niche cell populations including MSCs, pericytes and hematopoietic stem cells were maintained in relative quiescence for 7 days in proportions similar to that in freshly isolated cells. Colony-forming unit efficiency of enzymatically retrieved MSCs was significantly higher at day 14 compared to day 0; and in accordance with previously published works, it was fibroblast growth factor-2-dependant. CONCLUSIONS: Fibrin gels provide a simple, novel system in which to culture and study the complete fraction of bone marrow-derived mononuclear cells and may support the development of improved bone marrow cell-based therapies.

Microfabrication of Photo-Cross-Linked Hyaluronan Hydrogels by Single- and Two-Photon Tyramine Oxidation.

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.

Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production.

Evaluation of a press-fit osteochondral poly(ester-urethane) scaffold in a rabbit defect model.

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.