Population-based user-perceived experience of Rheumatic?: a novel digital symptom-checker in rheumatology.

OBJECTIVE: Digital symptom-checkers (SCs) have potential to improve rheumatology triage and reduce diagnostic delays. In addition to being accurate, SCs should be user friendly and meet patient's needs. Here, we examined usability and acceptance of Rheumatic?-a new and freely available online SC (currently with >44 000 users)-in a real-world setting. METHODS: Study participants were recruited from an ongoing prospective study, and included people ≥18 years with musculoskeletal complaints completing Rheumatic? online. The user experience survey comprised five usability and acceptability questions (11-point rating scale), and an open-ended question regarding improvement of Rheumatic? Data were analysed in R using t-test or Wilcoxon rank test (group comparisons), or linear regression (continuous variables). RESULTS: A total of 12 712 people completed the user experience survey. The study population had a normal age distribution, with a peak at 50-59 years, and 78% women. A majority found Rheumatic? useful (78%), thought the questionnaire gave them an opportunity to describe their complaints well (76%), and would recommend Rheumatic? to friends and other patients (74%). Main shortcoming was that 36% thought there were too many questions. Still, 39% suggested more detailed questions, and only 2% suggested a reduction of questions. CONCLUSION: Based on real-world data from the largest user evaluation study of a digital SC in rheumatology, we conclude that Rheumatic? is well accepted by women and men with rheumatic complaints, in all investigated age groups. Wide-scale adoption of Rheumatic?, therefore, seems feasible, with promising scientific and clinical implications on the horizon.

TNF overexpression and dexamethasone treatment impair chondrogenesis and bone growth in an additive manner.

Children with chronic inflammation are often treated with glucocorticoids (GCs) and many of them experience growth retardation. It is poorly understood how GCs interact with inflammatory cytokines causing growth failure as earlier experimental studies have been performed in healthy animals. To address this gap of knowledge, we used a transgenic mouse model where human TNF is overexpressed (huTNFTg) leading to chronic polyarthritis starting from the first week of age. Our results showed that femur bone length and growth plate height were significantly decreased in huTNFTg mice compared to wild type animals. In the growth plates of huTNFTg mice, increased apoptosis, suppressed Indian hedgehog, decreased hypertrophy, and disorganized chondrocyte columns were observed. Interestingly, the GC dexamethasone further impaired bone growth, accelerated chondrocyte apoptosis and reduced the number of chondrocyte columns in huTNFTg mice. We conclude that TNF and dexamethasone separately suppress chondrogenesis and bone growth when studied in an animal model of chronic inflammation. Our data give a possible mechanistic explanation to the commonly observed growth retardation in children with chronic inflammatory diseases treated with GCs.

Transcriptomic Profiling Reveals That HMGB1 Induces Macrophage Polarization Different from Classical M1.

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. In different conditions, macrophage polarization can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity via the Toll-like receptor (TLR) 4, the receptor for advanced glycation end products (RAGE), and C-X-C chemokine receptor (CXCR) 4. This study investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) stimulated with different HMGB1 redox isoforms using bulk RNA sequencing (RNA-Seq). Disulfide HMGB1 (dsHMGB1)-stimulated BMDMs showed a similar but distinct transcriptomic profile to LPS/IFNγ- and LPS-stimulated BMDMs. Fully reduced HMGB1 (frHMGB1) did not induce any significant transcriptomic change. Interestingly, compared to LPS/IFNγ- and LPS-, dsHMGB1-stimulated BMDMs showed lipid metabolism and foam cell differentiation gene set enrichment, and oil red O staining revealed that both dsHMGB1 and frHMGB1 alleviated oxidized low-density lipoprotein (oxLDL)-induced foam cells formation. Overall, this work, for the first time, used transcriptomic analysis by RNA-Seq to investigate the impact of HMGB1 stimulation on BMDM polarization. Our results demonstrated that dsHMGB1 and frHMGB1 induced distinct BMDM polarization phenotypes compared to LPS/IFNγ- and LPS- induced phenotypes.

Immunoprofiling of active and inactive systemic juvenile idiopathic arthritis reveals distinct biomarkers: a single-center study.

BACKGROUND: This study aimed to perform an immunoprofiling of systemic juvenile idiopathic arthritis (sJIA) in order to define biomarkers of clinical use as well as reveal new immune mechanisms. METHODS: Immunoprofiling of plasma samples from a clinically well-described cohort consisting of 21 sJIA patients as well as 60 age and sex matched healthy controls, was performed by a highly sensitive proteomic immunoassay. Based on the biomarkers being significantly up- or down-regulated in cross-sectional and paired analysis, related canonical pathways and cellular functions were explored by Ingenuity Pathway Analysis (IPA). RESULTS: The well-studied sJIA biomarkers, IL6, IL18 and S100A12, were confirmed to be increased during active sJIA as compared to healthy controls. IL18 was the only factor found to be increased during inactive sJIA as compared to healthy controls. Novel factors, including CASP8, CCL23, CD6, CXCL1, CXCL11, CXCL5, EIF4EBP1, KITLG, MMP1, OSM, SIRT2, SULT1A1 and TNFSF11, were found to be differentially expressed in active and/or inactive sJIA and healthy controls. No significant pathway activation could be predicted based on the limited factor input to the IPA. High Mobility Group Box 1 (HMGB1), a damage associated molecular pattern being involved in a series of inflammatory diseases, was determined to be higher in active sJIA than inactive sJIA. CONCLUSIONS: We could identify a novel set of biomarkers distinguishing active sJIA from inactive sJIA or healthy controls. Our findings enable a better understanding of the immune mechanisms active in sJIA and aid the development of future diagnostic and therapeutic strategies.

Disulfide and Fully Reduced HMGB1 Induce Different Macrophage Polarization and Migration Patterns.

Macrophage plasticity enables cells to obtain different functions over a broad proinflammatory and repairing spectrum. In different conditions, macrophages can be induced by high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein that activates innate immunity, to polarize towards a pro- (M1) or anti-inflammatory (M2) phenotype. In this study, we investigated the phenotypes of murine bone-marrow-derived macrophages (BMDMs) induced by different HMGB1 redox isoforms in depth. Our results demonstrate that disulfide HMGB1 (dsHMGB1) induces a unique macrophage phenotype that secretes pro-inflammatory cytokines, rather than inducing metabolic changes leading to nitric oxide production. Fully reduced HMGB1 (frHMGB1) did not induce macrophage polarization. The migrating function of BMDMs was measured by scratch assay after the stimulation with dsHMGB1 and frHMGB1. Both dsHMGB1 and frHMGB1 induced cell migration. We found that dsHMGB1 mediates cytokine secretion and cellular motility, mainly through toll-like receptor 4 (TLR4). Importantly, our data shows that dsHMGB1 and frHMGB1 induce distinct BMDM polarization phenotypes, and that dsHMGB1 induces a unique phenotype differing from the classical proinflammatory macrophage phenotype.

Pain behaviour assessments by gait and weight bearing in surgically induced osteoarthritis and inflammatory arthritis.

OBJECTIVE: . Osteoarthritis (OA) is the most common cause of joint pain. Animal models and relevant assays for measurement of pain-related behaviours are important tools for studies of mechanisms inducing and sustaining pain in OA. The aim of this study was to evaluate two different assessments of weight bearing; stationary and during locomotion, and to explore their feasibility to detect analgesic effects in vivo. Two fundamentally different mouse models of joint arthritis were investigated; surgical transection of the anterior cruciate ligament (ACLT) resulting in destabilization of the joint with subsequent structural deterioration resembling OA, and monoarthritis induced by injection of Complete Freund´s Adjuvant (CFA) into the ankle joint capsule. DESIGN: . Mice were subjected to ACLT or CFA injection into the ankle joint. Stationary weight bearing was performed up to twenty weeks after ACLT, and for two weeks after CFA. In addition, mice with CFA-induced monoarthritis were assessed for gait and weight bearing during locomotion, and the effects of an anti-NGF antibody (MEDI578) were tested. End point histopathological analysis was performed in knee joints of ACLT mice, and in mice with ankle joint injection of CFA at eight days after injection. RESULTS: . Both the surgical ACLT and CFA-induced monoarthritis reduced stationary weight bearing on the affected paw. The reduction in weight bearing was compensated by all other legs, but differently when stationary compared to during locomotion in the CFA-injected mice. The behavioural effects of ACLT correlated to the structural changes of the joint. In the CFA-induced monoarthritis, showing a massive infiltration of inflammatory cells at 8 days, MEDI578 significantly attenuated the pain-like behaviours. CONCLUSIONS: . The pain-like behaviour detected is mainly due to inflammation and not to the same degree to structural changes in the joint. Behavioural effects after ACLT were too small for pharmacological evaluation of pain relief. In contrast, the inflammation after CFA injection caused a long-lasting effect on pain-like behaviours such as weight bearing and gait, which could be attenuated by administration of an anti NGF antibody.

Humanin prevents undesired apoptosis of chondrocytes without interfering with the anti-inflammatory effect of dexamethasone in collagen-induced arthritis.

OBJECTIVES: Prolonged use of glucocorticoids (GCs) for treatment of inflammatory and autoimmune conditions may have several negative side effects, such as impaired bone growth which has been linked to increased apoptosis in growth plate chondrocytes. It has recently been shown that humanin, a small mitochondrial derived peptide, rescues growth plate chondrocytes from GC-induced apoptosis. Our aim was to study if a synthetic analogue of humanin, [Gly14]-HNG (HNG), can be safely used to prevent GC-induced toxicity in growth plate chondrocytes without interfering with the desired anti-inflammatory effect in an in vivo model of arthritis. METHODS: Arthritis was induced in DBA/1 mice by collagen type II in complete Freund's adjuvant and the animals were treated with Dexamethasone (Dexa) (0.25 mg/kg/day) with or without HNG (100 µg/kg/day) for 14 days. The animals were observed daily for the presence of arthritis including signs of erythema and swelling of the joints. The paws were scored based on the severity of the swelling. After termination, histological scoring was performed of all paws. Chondrocyte apoptosis and proliferation were analysed by TUNEL assay and PCNA staining, respectively. RESULTS: We found that HNG treatment in combination with Dexa protected from Dexa-induced chondrocyte apoptosis in both articular and growth plate cartilage. Furthermore, based on clinical and histology scoring analyses, HNG did not interfere with the desired anti-inflammatory effect of Dexa. CONCLUSIONS: Our results suggest that the combination of HNG and GCs may provide a new treatment strategy in conditions of chronic inflammation, which could potentially prevent bone growth impairment.

Juvenile idiopathic arthritis patients have a distinct cartilage and bone biomarker profile that differs from healthy and knee-injured children.

OBJECTIVES: Joint destruction is a hallmark of juvenile idiopathic arthritis (JIA). Clinical evaluation and radiographic imaging are current methods to identify destruction. Biomarkers could aid an earlier and more sensitive diagnosis. Our aim was to investigate levels of bone and cartilage degradation biomarkers in JIA patients, compared to healthy children or juveniles with knee injuries. METHODS: Triple-paired synovial fluid, plasma and urine samples from 29 JIA patients were compared to 61 plasma samples from healthy children and synovial fluid from 41 knee-injured juveniles. Cartilage biomarkers ARGS neoepitope of aggrecan (ARGS), cartilage oligomeric matrix protein (COMP), type II collagen epitope (C2C), bone biomarkers N-terminal type I collagen cross-linked telopeptide (NTX-I) and tartrate-resistant acid phosphatase 5b (TRAP5b) were analysed by immunoassays. RESULTS: Plasma levels of ARGS, C2C, COMP and TRAP5b were increased in JIA compared to healthy children. Compared to knee-injured juveniles, synovial fluid C2C and TRAP5b were increased in JIA, while ARGS and COMP were decreased. For JIA patients, local (synovial fluid) and systemic (plasma/urine) levels of bone biomarkers correlated positively; age correlated negatively to plasma levels of C2C and TRAP5b; no correlation was found between biomarkers and gender, affected joint count, disease duration or medication. CONCLUSIONS: Elevated levels of destruction biomarkers in JIA compared to healthy children indicate a potential to serve as clinical tools for destructive joint disease. High levels of TRAP5b, NTX-I and collagen II in JIA in contrast to more pronounced aggrecan and COMP degradation in juvenile knee injuries, suggests that JIA patients have a unique biomarker pattern, different from healthy and knee-injured children.

Fragmented hyaluronan has no alarmin function assessed in arthritis synovial fibroblast and chondrocyte cultures.

Hyaluronan (HA) is a large polymer and an important component of the extracellular matrix. During homeostasis, high molecular mass HA is the predominant form, but upon inflammation, degradation products of HA accumulate. These HA fragments (HA-fs) have been reported to possess pro-inflammatory activities and thus act as alarmins, notifying immune cells of danger via TLR4 and CD44. HA is found in large quantities in synovial joint fluid. In order to reveal a potential role of HA-fs in arthritis pathogenesis, the in vitro effects of HA of various molecular masses (from 1680 kDa to oligosaccharide HA) on synovial fibroblasts and chondrocytes from rheumatoid arthritis patients, and on peripheral blood mononuclear cells from healthy donors, were investigated. TLR4 and CD44 surface expression was confirmed by immunocytochemistry, and cell activation was determined based on cytokine and chemokine production. While the cell types investigated expressed TLR4 and CD44, no increased release of IL-1ß, IL-6, IL-8, IL-10, IL-12 or TNF-α was detected after HA stimulation. Similarly, HA did not enhance activation after priming cells with low doses of LPS or by forming complexes with LPS. Hence, this study does not support the common view of HA-fs being pro-inflammatory mediators and it is not likely that HA-fs generated during arthritis contribute to disease pathogenesis.

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice.

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP-induced acute liver injury (APAP-ALI) and justifies development of anti-inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N-acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP-ALI. The anti-HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti-HMGB1 polyclonal antibody treatment improves survival in a model of APAP-ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti-HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP-ALI. The mouse anti-HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody-Fc backbones. Effector function-deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP-ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP-induced serum elevations of alanine aminotransferase and microRNA-122 and completely abrogated markers of APAP-induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C-X-C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. CONCLUSION: This is the first report describing the generation of a partly humanized HMGB1-neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP-ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1-specific therapy as a means to treat APAP-ALI and other inflammatory conditions. (Hepatology 2016;64:1699-1710).

Nitrosative modifications of the Ca2+ release complex and actin underlie arthritis-induced muscle weakness.

OBJECTIVE: Skeletal muscle weakness is a prominent clinical feature in patients with rheumatoid arthritis (RA), but the underlying mechanism(s) is unknown. Here we investigate the mechanisms behind arthritis-induced skeletal muscle weakness with special focus on the role of nitrosative stress on intracellular Ca(2+) handling and specific force production. METHODS: Nitric oxide synthase (NOS) expression, degree of nitrosative stress and composition of the major intracellular Ca(2+) release channel (ryanodine receptor 1, RyR1) complex were measured in muscle. Changes in cytosolic free Ca(2+) concentration ([Ca(2+)]i) and force production were assessed in single-muscle fibres and isolated myofibrils using atomic force cantilevers. RESULTS: The total neuronal NOS (nNOS) levels were increased in muscles both from collagen-induced arthritis (CIA) mice and patients with RA. The nNOS associated with RyR1 was increased and accompanied by increased [Ca(2+)]i during contractions of muscles from CIA mice. A marker of peroxynitrite-derived nitrosative stress (3-nitrotyrosine, 3-NT) was increased on the RyR1 complex and on actin of muscles from CIA mice. Despite increased [Ca(2+)]i, individual CIA muscle fibres were weaker than in healthy controls, that is, force per cross-sectional area was decreased. Furthermore, force and kinetics were impaired in CIA myofibrils, hence actin and myosin showed decreased ability to interact, which could be a result of increased 3-NT content on actin. CONCLUSIONS: Arthritis-induced muscle weakness is linked to nitrosative modifications of the RyR1 protein complex and actin, which are driven by increased nNOS associated with RyR1 and progressively increasing Ca(2+) activation.

In situ cross-linkable hyaluronan hydrogel enhances chondrogenesis.

The present work describes the feasibility of a cross-linkable injectable hyaluronan hydrogel for cartilage repair. The hydrogel used is a two-component system based on aldehyde-modified hyaluronan and hydrazide-modified polyvinyl alcohol, which are rapidly cross-linked in situ upon mixing. The in vitro study showed that chondrocytes and mesenchymal cells cultured in the gel form cartilage-like tissue, rich in glycosaminoglycans, collagen type II and aggrecan. In a rabbit animal model the injection of the hydrogel improved the healing of a full-thickness cartilage defect created in the knee as compared to non-treated controls. This rabbit study showed that the regenerated cartilage defects stained more intensely for type II collagen upon treatment with the hydrogel. The hyaluronan-based hydrogel may be used as a delivery vehicle for both growth factors and/or cells for cartilage repair. The in vivo study also indicated that the hydrogel alone has a beneficial effect on cartilage regeneration.

Extracellular matrix-polymer hybrid materials produced in a pulsed-flow bioreactor system.

Cell adhesion, interaction with material, cell proliferation and the production of an extracellular matrix (ECM) are all important factors determining the successful performance of an engineered scaffold. Scaffold design should aim at creating structures which can guide cells into forming new, functional tissue. In this study, the concept of in situ deposition of ECM by human dermal fibroblasts onto a compliant, knitted poly (ethyleneterephtalate) support is demonstrated, creating in vitro produced ECM polymer hybrid materials for tissue engineering. Comparison of cells cultured under static and dynamic conditions were examined, and the structure and morphology of the materials so formed were evaluated, along with the amount collagen deposited by the seeded cells. In vitro produced ECM polymer hybrid scaffolds could be created in this way, with the dynamic culture conditions increasing ECM deposition. Histological analysis indicated a homogenous distribution of cells in the 1 mm thick scaffold, surrounded by a matrix-like structure. ECM deposition was observed throughout the materials wigh 81.6 microg/cm(2) of collagen deposited after 6 weeks. Cell produced bundles of ECM fibres bridged the polymer filaments and anchored cells to the support. These findings open hereto unknown possibilities of producing materials with structure designed by engineering together with biochemical composition given by cells.

A poly(lactic acid-co-caprolactone)-collagen hybrid for tissue engineering applications.

A biodegradable hybrid scaffold consisting of a synthetic polymer, poly(lactic acid-co-caprolactone) (PLACL), and a naturally derived polymer, collagen, was constructed by plastic compressing hyperhydrated collagen gels onto a flat warp-knitted PLACL mesh. The collagen compaction process was characterized, and it was found that the duration, rather than the applied load under the test conditions in the plastic compression, was the determining factor of the collagen and cell density in the cell-carrying component. Cells were spatially distributed in three different setups and statically cultured for a period of 7 days. Short-term biocompatibility of the hybrid construct was quantitatively assessed with AlamarBlue and qualitatively with fluorescence staining and confocal microscopy. No significant cell death was observed after the plastic compression of the interstitial equivalents, confirming previous reports of good cell viability retention. The interstitial, epithelial, and composite tissue equivalents showed no macroscopic signs of contraction and good cell proliferation with a two- to threefold increase in cell number over 7 days. Quantitative analysis showed a homogenous cell distribution and good biocompatibility. The results indicate that viable and proliferating multilayered tissue equivalents can be engineered using the PLACL-collagen hybrid construct in the space of several hours.

Bulk collagen incorporation rates into knitted stiff fibre polymer in tissue-engineered scaffolds: the rate-limiting step.

Fabrication of tissue-engineered constructs in vitro relies on sufficient synthesis of extracellular matrix (ECM) by cells to form a material suitable for normal function in vivo. Collagen synthesis by human dermal fibroblasts grown in vitro on two polymers, polyethylene terephthalate (PET) and polyglycolic acid (PGA), was measured by high-performance liquid chromatography (HPLC). Cells were either cultured in a dynamic environment, where meshes were loaded onto a pulsing tube in a bioreactor, or in a static environment without pulsing. Collagen synthesis by cells cultured on a static mesh increased by six-fold compared to monolayer culture, and increased by up to a further 5.4-fold in a pulsed bioreactor. However, little of the collagen synthesized was deposited onto the meshes, almost all being lost to the medium. The amount of collagen deposited onto meshes was highest when cells were cultured dynamically on PET meshes (17.6 microg), but deposition still represented only 1.4% of the total synthesized. Although total collagen synthesis was increased by the use of 3D culture and the introduction of pulsing, the results suggest that the limiting factor for fabrication of a tissue-engineered construct within practical timescales is not the amount of collagen synthesized but the quantity retained (i.e. deposited) within the construct during culture. This may be enhanced by systems which promote or assemble true 3D multi-layers of cells.

In vitro degradation and in vivo biocompatibility study of a new linear poly(urethane urea).

Segmented poly(urethane urea)s (PUUs) with hard segments derived only from methyl 2,6-diisocyantohexanoate (LDI) without the use of a chain extender have previously been described. These materials, which contain hard segments with multiple urea linkages, show exceptionally high strain capability (1600-4700%). In the study reported here, the rate and effect of hydrolysis of these materials were determined for gamma-sterilized and nonsterilized samples. Materials investigated contained PCL, PTMC, P(TMC-co-CL), P(CL-co-DLLA), or P(TMC-co-DLLA) as soft segments and, as well as their mechanical properties, changes in mass, inherent viscosity (I.V.), and thermal properties were studied over 20 weeks. Results showed that the degradation rate was dependant on the soft segment structure, with a higher rate of degradation for the polyester-dominating PUUs exhibiting a substantial loss in I.V. A tendency of reduction of tensile strength and strain hardening was seen for all samples. Also, loss in elongation at break was detected, for PUU-P(CL-DLLA) it went from 1600% to 830% in 10 weeks. Gamma radiation caused an initial loss in I.V. and induced more rapid hydrolysis compared with nonsterilized samples, except for PUU-PTMC. A cytotoxicity test using human fibroblasts demonstrated that the material supports cell viability. In addition, an in vivo biocompatibility study showed a typical foreign body reaction after 1 and 6 weeks.

Polarized protein membrane for high cell seeding efficiency.

A new type of scaffold for tissue engineering was developed to give enhanced cell seeding in three dimensions. A gradient of either collagen or fibrin protein was prepared, supported by a knitted poly(ethylene terephtalate) PET fabric. The membranes were, after hydrolysis and acetic acid wash, submerged in a protein solution for adsorption followed by immersion into a gelling agent. The immediate contact between the protein solution held by the fabric and the gelling agent resulted in a dense, fibrous protein network with pore sizes around 0.5 microm at the surface, and larger pores of 10-50 microm size throughout the interior of the fabric as observed by scanning electron microscopy. By separating the fabric double layers holding this network, a gradient porosity membrane was produced. To evaluate the fractions of cells trapped in the matrix upon seeding, i.e. the seeding efficiency, 500 microl 3T3 fibroblasts cell suspension containing one million cells was seeded by filtering through the gradient protein membrane. For both the collagen and fibrin membranes, the seeding efficiency was approximately 93%, which was significantly higher than that of 28% from the corresponding PET fabric without protein immobilization. Attempt to seed cells from the dense side of the protein networks resulted in no cell penetration into the scaffold. Histology on subsequent culture of the cells in the scaffold demonstrated viability and proliferation in three dimensions throughout the scaffold. This new and simple way of producing scaffolds play an important role when the cells are precious or scarce and cell seeding in three dimensions is important.