Intervertebral disc (IVD) degeneration is inadequately understood due to the lack of in vitro systems that fully mimic the mechanical and biological complexity of this organ. We have recently made an advancement by developing a bioreactor able to simulate physiological, multiaxial IVD loading and maintain the biological environment in ex vivo IVD models [1]. To validate this new bioreactor system, we simulated natural spine movement by loading 12 bovine IVDs under a combination of static compression (0.1 MPa), cyclic flexion/extension (±3˚, ±6˚ or 0-6˚) and cyclic torsion (±2˚, ±4˚ or 0-4˚) for more than 10’000 (0.2 Hz) or 100’000 (1 Hz) cycles over 14 days. A higher number of cycles increased the release of glycosaminoglycans and nitric oxide, as an inflammation marker, whereas fewer cycles maintained these two factors at physiological levels. All applied protocols upregulated the expression of MMP13 in the outermost annulus fibrosus (AF), indicating a collagen degradation response. This was supported by fissures observed in the AF after a longer loading duration. Increasing loading cycles induced high cell death in the nucleus pulposus and inner AF, while with fewer cycles, high cell viability was maintained in all IVD regions, irrespective of the magnitude of rotation. Less frequent multiaxial loading maintains IVD homeostasis while more frequent loading initiates an IVD degenerative profile. Specifically, the morphological and molecular changes were localized in the AF, which can be associated with combined flexion/extension and torsion. More loading cycles induced region-specific cell death and a higher release of extracellular matrix molecules from the innermost IVD regions, likely associated with longer exposure to static compression. Altogether, we demonstrated the advantages of the multiaxial bioreactor to study region-specific response in the IVD, which will allow a more profound investigation of IVD degeneration under different combinations of motions

Introduction. Supraspinatus tears comprise most rotator cuff injuries, the leading cause of shoulder pain and an increasing problem with ageing populations. Surgical repair of considerable or persistent damages is customary, although not invariably successful. Tissue engineering presents a promising alternative to generate functional tissue constructs with improved healing capacities. This study explores tendon tissue constructs’ culture in a platform providing physiological mechanical stimulation and reports on the effect of different loading regimes on the viability of human tendon cells. Method. Porcine decellularized tendon scaffolds were fixed into flexible, self-contained bioreactor chambers, seeded with human tenocytes, allocated in triplicates to either static control, low (15±0.8Newtons [N]), medium (26±0.5N), or high (49±2.1N)-force-regime groups, connected to a perfusion system and cultured under standard conditions. A humanoid robotic arm provided 30-minute adduction/abduction stimulation to chambers daily over a week. A metabolic activity assay served to assess cell viability at four time points. Statistical significance = p<0.05. Result. One day after beginning mechanical stimulation, chambers in the medium and high-force regimes displayed a rise in metabolic activity by 3% and 5%, respectively. By the last experimental day, all mechanical stimulation regimes had induced an augment in cell viability (15%, 57% and 39% with low, medium, and high loads, respectively) matched against the static controls. Compared to all other conditions, the medium-force regime prompted an increased relative change in metabolic activity for every time point set against day one (p<0.05). Conclusion. Human tenocytes’ viability reflected by metabolic activity in a physiologically relevant bioreactor system is enhanced by loading forces around 25N when mechanically stimulating using adduction/abduction motions. Knowing the most favourable load regime to stimulate tenocyte growth has informed the ongoing exploration of the distinctive effect of different motions on tendon regeneration towards engineering tissue grafts. This work was supported by the Engineering and Physical Sciences Research Council EP/S003509/1

The success of cementless orthopaedic implants relies on bony ingrowth and active bone remodelling. Much research effort is invested to develop implants with controllable surface roughness and internal porous architectures that encourage these biological processes. Evaluation of these implants requires long-term and costly animal studies, which do not always yield the desired outcome requiring iteration. The aim of our study is to develop a cost-effective method to prescreen design parameters prior to animal trials to streamline implant development and reduce live animal testing burden. Ex vivo porcine cancellous bone cylinders (n=6, Ø20×12mm) were extracted from porcine knee joints with a computer-numerically-controlled milling machine under sterile conditions within 4 hours of animal sacrifice. The bone discs were implanted with Ø6×12mm additive manufactured porous titanium implants and were then cultured for 21days. Half underwent static culture in medium (DMEM, 10% FBS, 1% antibiotics) at 37°C and 5% CO. 2. The rest were cultured in novel high-throughput stacked configuration in a bioreactor that simulated physiological conditions after surgery: the fluid flow and cyclic compression force were set at 10ml/min and 10–150 N (1Hz,5000 cycles/day) respectively. Stains were administered at days 7 and 14. Samples were evaluated with widefield microscopy, scanning electron microscopy (SEM) and with histology. More bone remodelling was observed on the samples cultured within the bioreactor: widefield imaging showed more remodelling at the boundaries between the implant-bone interface, while SEM revealed immature bone tissue integration within the pores of the implant. Histological analysis confirmed these results, with many more trabecular struts with new osteoid formation on the samples cultured dynamically compared to static ones. Ex vivo bone can be used to analyse new implant technologies with lower cost and ethical impact than animal trial. Physiological conditions (load and fluid flow) promoted bone ingrowth and remodelling

Introduction Tissue engineering aims to produce a cellular structure in an extracellular matrix, which when implanted heals tissue defects. To tissue-engineer bone suitable cells need to be grown on a scaffold. In this study we grew human marrow cells as they can differentiate into osteoblasts, on porous hydroxyapatite (HA) scaffolds, as this is osteoconductive, allows cell penetration and in growth of capillaries after implantation. Increased extravascular perfusion through bone increases new bone formation. So we reproduced these physiological conditions in our novel bioreactor by perfusing scaffolds at 6ml/hr. Hypotheses 1. Culture in our bioreactor improved cell penetration through HA scaffolds compared to static conditions. 2. Human mesenchymal stem cells (MSCs) cultured in our bioreactor differentiated into osteo-blasts and produced bone extracellular matrix. Method MSCs were isolated from 8 human bone marrow aspirates taken from patients following informed consent. For each experiment 16 scaffolds were seeded with MSCs and comparisons were made between the two conditions. After 7 days culture the scaffolds were sectioned longitudinally and the number of cells at increasing depths were counted. The scaffolds were observed under SEM & TEM. Osteoblastic markers ALP and type I pro-collagen (PICP) were measured. Results Penetration of cells through the scaffolds was significantly greater when cultured in the bioreactor. After 14 days in bioreactor culture the HA was covered with cuboidal cells, consistent with osteoblasts, however in static culture cells remained fibroblastic. TEM results showed that MSCs in the bioreactor produced organised collagen matrix after 21 days and osteoid by 28 days, but no collagen matrix was observed following static culture. ALP and PICP were significantly greater over 15 days culture when in our bioreactor. Conclusions These results show that when MSCs were cultured in our bioreactor they attached and penetrated through porous HA scaffolds, whereas in static conditions few cells penetrated below 2mm. Our bioreactor significantly improved 3-dimensional growth, resembling tissue. Moreover, MSCs grown on HA in the bioreactor produced significantly more ALP and PICP indicating osteoblastic differentiation. Furthermore, bone osteoid was produced. Therefore this culture method could be use to convert autologous MSCs from human marrow into tissue-engineered bone which could be used to heal defects after tumor excision

Joint pain, as a consequence of cartilage degeneration or trauma results in severe pain or disability for millions of individuals worldwide. However, the potential for cartilage to regenerate is limited and there is an absence of clinically viable cartilage formation regimes. Cartilage is composed of only one cell type, is avascular and has a relatively simple composition and structure, thus cartilage tissue engineering has tremendous potential. Therefore, to address this clinical need, we have adopted a tissue engineering approach to the generation of cartilage ex vivo from mesenchymal cell populations encapsulated in polysaccharide templates form alginate and chitosan that favours chondrogenesis, and cultured within perfused or rotating bioreactor systems. To drive the chondrogenic phenotype, alginate beads were encapsulated with isolated human bone marrow cells, human articular chondrocytes or a combination of both in a 2:1 ratio, with the addition of TGF-â3, and placed in either a Synthecon rotating-wall bioreactor, perfused at a flow rate of 1ml/hour, or held in static conditions for 28 days. Alcian Blue and Sirius Red staining indicated ordered, structured and even cell distribution within capsules from the rotating bioreactor system in comparison with perfused and static conditions. Furthermore, alginate beads encapsulated with mixed cell populations that were cultured under static and rotating-wall conditions revealed positive staining for both collagen and proteoglycan, and with areas that closely resembled the formation of osteoid. Cell viability, assessed using the fluorescent dye Cell Tracker Green, indicated a higher proportion of metabolically active cells in capsules from the rotating-wall bioreactor than perfused or static under the conditions examined. Immunohistochemistry indicated the expression of type II collagen, SOX9 and C-MYC in samples from all conditions after 28 days. C-MYC is implicated in cell proliferation and differentiation and type II collagen and SOX9 are cartilage-specific markers. Biochemical analysis revealed significantly increased (p < 0.05) protein in samples encapsulated with mixed cell populations compared with alginate samples that were encapsulated with either bone marrow or chondrocytes. There was also a significant increase in protein in all samples that were cultured in the rotating-wall bioreactor in comparison with perfused or static conditions after 28 days. A significant increase in DNA was observed in the rotating-wall than perfused or static for the bone marrow cultures. Interestingly in chondrocyte cultures perfused conditions were found to result in significantly higher DNA than rotating-wall and static, and static conditions resulted in significantly higher DNA for alginate encapsulated with mixed cell populations. The current studies outline a tissue engineering approach utilising progenitor populations, bioreactors and appropriate stimuli to promote the formation of cartilage within a unique innovative polysaccharide capsule structure, and indicate the potential of rotating-wall systems to promote cartilage formation. Understanding the conditions required for the generation of functional cartilage constructs using such bioreactor systems carries significant clinical potential

Bioreactors have been used in articular cartilage tissue engineering (AC-TE) to apply different mechanical stimuli in an attempt to better mimic the native AC microenvironment. However, these systems are often highly complex, costly and not very versatile. In this work, we propose a simple and customizable perfusion bioreactor fabricated by 3D-extrusion to study the effect of shear stress in human bone-marrow mesenchymal stem cells (hBMSC) cultured in 3D porous polycaprolactone (PCL) scaffolds. Prototype models were designed in a CAD-software to perfectly fit the scaffolds and computational fluid dynamics analysis was used to predict the fluid velocities and shear stress forces inside the bioreactor. For the culture studies, hBMSC-PCL constructs were cultured under static expansion for 2 weeks and then transferred to the ABS-extruded bioreactors for continuous perfusion culture (0.2mL/min) under chondrogenic induction for additional 3 weeks. Perfused constructs showed similar cell proliferation and higher sGAG production in comparison to the static counterparts (bioreactor without perfusion). Constructs exposed to shear stress stimuli presented higher expressions of chondrogenic genes (COLII/Sox9/Aggrecan) and reduced expressions of COLI and Runx2 (osteogenic) than static group. However, the higher expression of COLX in the perfused constructs suggests a shear stress role in AC hypertrophy. Both conditions (perfused/static) stained positively for GAG deposition and for the presence of collagen II and aggrecan. Overall, the results provide a proof-of-concept of our customizable extruded bioreactor envisaging applications as a platform for AC-TE repair strategies and in the development of more reliable in vitro models for disease modelling and drug screening

Introduction. The combined incubation of a composite scaffold with bone marrow stromal cells in a perfusion bioreactor could make up a novel hybrid graft material with optimal properties for early fixation of implant to bone. The aim of this study was to create a bioreactor activated graft (BAG) material, which could induce early implant fixation similar to that of allograft. Two porous scaffold materials incubated with cells in a perfusion bioreactor were tested in this study. Methods and Materials. Two groups of 8 skeletally mature female sheep were anaesthetized before aspiration of bone marrow from the iliac crest. For both groups, mononuclear cells were isolated, and injected into a perfusion bioreactor (Millenium Biologix AG, Switzerland). Scaffold granules Ø∼900–1500 μm, ∼88% porosity) in group 1, consisted of hydroxyapatite (HA, 70%) with -tricalcium-phosphate (−TCP, 30%) (Danish Technological Institute, Denmark). The granules were coated with poly-lactic acid (PLA) 12%, in order to increase the mechanical strength of the material (Phusis, France). Scaffold granules Ø∼900–1400 μm, 80% porosity) in group 2 consisted of pure HA/-TCP (Fin Ceramica, Italy). For both groups, cells were incubated in the bioreactor for 2 weeks. Fresh culture medium supplemented with dexamethasone and ascorbic-acid was added every third or fourth day. Porous titanium alloy implants with diameter=length=10mm (Biomet, USA) were inserted bilaterally in each of the distal femurs of the sheep; thus 4 implants in each sheep. The concentric gap (2 mm) surrounding the implant was filled with 1) BAG (autogenous), 2) granules, 3) granules+bone marrow aspirate (BMA, autologous) or 4) allograft. The sheep were euthanized after 6 weeks. Distal femurs were removed and implant-bone samples were divided in two parts. The superficial part was used for mechanical testing and micro-CT scanning, and the profound part for histomorphometry. Push-out tests were performed on an 858 Bionix MTS hydraulic materials testing machine. Shear mechanical properties between implant and newly generated bone were calculated to assess implant fixation. Results were assessed by One-way ANOVA. P-values less than 0.05 were considered significant. Results. One sheep in group 1 had to be euthanized after 4 weeks (excluded). One implant in each group was loosened and could not undergo push-out test (excluded). Group 1: No significant differences regarding failure energy (kJ/m2, p=0.44) or ultimate shear strength (MPa, p=0.17) could be seen. Shear stiffness (MPa) was significantly higher for the allograft group (p=0.04). Group 2: No significant differences regarding failure energy (p=0.11) or shear stiffness (p=0.52) could be seen. Ultimate shear strength was significantly higher for allograft (p=0.04). Results from μ-CT scanning and histomorphometry are pending. Discussion and Conclusion. The present study shows a possible effect of bioreactor activated bone substitute on early implant fixation. We are currently working on bone microarchitecture surrounding implant and histomorphometry. These results will aid in determining if BAG could make up a promising alternative for allograft as bone graft material

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the engineered cartilage grafts so far proposed have successfully shown to mimic the morphological and biochemical appearance of hyaline cartilage, they are generally mechanically inferior to the natural tissue. 1. In this study a new bioreactor device was realized to test innovative scaffolds under physiological stimulation (i.e. perfusion fluid flow and dynamic compression), with the aim to produce a more functional engineered tissue construct for articular applications. The computer-controlled bioreactor system has been properly designed to simultaneously provide static or dynamic compression and/or continuous perfusion to 3D engineered constructs, reproducing the physiological loads to which the articular cartilage is subjected. The specifically designed bioreactor comprises a chamber where the grafts are accommodated, a porous piston connected to a linear stepper motor (Dings, Model 34-2080-4-300), which controls its movement to provide mechanical stimulation and a peristaltic pump (Watson-Marlow, Model 323S), connected by joints and pipes to the culture chamber to ensure a continuous media perfusion. As piston for compression, a sintered stainless steel filter (43% of porosity) was adopted to allow the perfusion of the culture media during physical stimulation. The culture chamber is composed by a hollow cylinder (30 mm × 40,5 mm) and a base realized as a single object. They are made in polycarbonate for its characteristics of transparency and infrangibility and linked to a Nylon cover through four brass tie rods unscrewable from above. The chamber has been designed to accommodate simultaneously different constructs of any size and shape and stimulate them with perfusion and/or dynamic compression. A finites elements program was used to mimic the effects of perfusion and compression regime on the scaffolds cultured within the bioreactor chamber. The bioreactor was properly designed and developed. Particular attention has been paid to the implementation of a simple, compact and economical system. It was then tested by using different polymeric porous scaffolds (PVA, collagen, Gelatin grafts, both porous and not) cultured with mesenchymal cells up to two weeks. The system has been validated in terms of sterility, experimental reproducibility and ease to use. The structural stability of grafts over time has been observed; moreover cells adhesion, proliferation and matrix production under different chemical-physical stimuli conditions is under investigation. We have realized a novel bioreactor system representing an artificial articular niche, where a dynamic compression combined with fluid perfusion allows to functionally and mechanically validate tissue substitutes, besides investigating the response of engineered cartilaginous tissues to physical stimuli mimicking the natural cartilage micro-environment. Such bioreactor may be in fact adopted as a sort of articular simulator for promoting and standardizing the new tissue formation in vitro, preconditioning cell fate through the application of proper artificial stimuli. Moreover, they can be valid tools to investigate physiological processes and novel therapeutic approaches avoiding controversial animal models

Mechanical loading regulates the metabolism of chondrocytes in cartilage1. Nowadays, studies exploring the in vitro response of cartilage towards loading often rely on bioreactor experiments applying only compressive loading. This is likely not sufficiently representative for the complex multi-directional loading profile in vivo (i.e. where typical compressive and shear loading are both present). The impact of multi-axial loading is specifically relevant in the context of the onset of osteoarthritis (OA) due to joint destabilization. Here, alterations in the 3D loading profile, and in particular increased shear forces, are suggested to initiate catabolic molecular responses leading to cartilage degeneration3. However, in vitro/ex vivo data confirming this hypothesis are currently lacking. Therefore, we aim to investigate how increased shear loading affects the metabolism and ECM deposition of a healthy chondrogenic cell line and if this response is different in osteoarthritic primary chondrocytes. A murine chondrogenic precursor cell line (ATDC5) and primary human osteoarthritic articular chondrocytes (hOACs) were encapsulated in 2.2% alginate disks and cultured in DMEM medium for three days. Hydrogels seeded with the different cell groups were loaded in the TA ElectroForce BioDynamic Bioreactor and subjected to following loading conditions: (a) 10% compression at 1Hz for 1h, (b) 10% compression and 10° shear loading at 1Hz for 1h. Unloaded constructs were used as control. After loading, hydrogel constructs were stabilized in culture medium for 2 hours, to facilitate adequate gene expression responses, before being dissolved and snap frozen. RNA was isolated and gene expression levels specific for anabolic pathways, characterized by extracellular matrix (ECM) genes (Col2a1, Aggrecan and Perlecan), catabolic processes (MMP-3 and MMP-13) and chondrogenic transcription factor (Sox9) were evaluated using RT-qPCR. The TA ElectroForce BioDynamic Bioreactor was successfully set-up to mimic cartilage loading. In ATDC5 cells, compression elicits an increase in all measured ECM genes (Col2a1, Aggrecan and Perlecan) compared to unloaded controls, suggesting an anabolic response. This upregulation is decreased when adding additional shear strain. In contrast to ATDC5 cells, the anabolic response of proteoglycans Aggrecan and Perlecan to compressive loading was lower in osteoarthritic chondrocytes, and Col2a1 expression appeared decreased. Adding shear strain reversed this effect on Col2a1 expression. Multi-directional loading increased transcription factor Sox9 expression compared to compression in both ATDC5 and OA chondrocytes. In OA chondrocytes, both loading regimens increased MMP-3 and MMP-13 expression. Shear loading reduces the anabolic effect of compressive loading in both cell types. OA cells presented more catabolic response to mechanical loading compared to precursors, given the increase in catabolic enzymes MMP-3 and MMP-13

Introduction: Recently, co-transplantion of mesenchy-mal stem cells (MSCs) with hematopoietic stem cells (HSCs) has been shown to alleviate complications such as GVHD and speeding recovery of HSCs. This in vivo finding suggests that coculture of MSCs and HSCs may enhance their growth potentials in vitro. As the large-scale expansion of HSCs has been achieved by NASA’s suspension culture system, we further examined the effects of this suspension culture system (rotary bio-reactor) on MSCs’ proliferation and differentiation potentials in vitro. Methods: Mononuclear cell fractions (MNCs) of human bone marrow aspirates (n=6, ages 46–81) were collected by density gradient centrifugation. The cells were inoculated into bioreactor (RCCS, Synthecon Inc., Texas, USA) at the concentration 1x10. 6. cells/ml, in Myelocult. TM. medium supplemented with 50ng/ml SCF, 20ng/ ml rhIL-3 and rhIL-6 (10ng/ml SCF, 2ng/ml IL-3 and IL-6 after the first feeding) and 10-6 M hydrocortisone for 8 days. The medium was fully exchanged after 3 days and 20% daily thereafter. Total cell numbers in the bioreactor were counted daily using hemacytometer. Cells from day 1, 4, and 8 cultures were subjected to tri-color flow cytometry examination using CD34, CD44, and Stro-1 antibodies. By the end of 8 day culture, the output cells were resuspended in DMEM medium with 10% FBS and cultured in T75 flasks at 1x10. 5. cells/cm2 for further 3 weeks. Upon harvest, half of the attached MSCs were prepared for western blotting assay using various antibodies. The other half was further cultured for 13–28 days in osteogenic, chondrogenic, and adipogenic induction medium respectively. Cell differentiation results were examined by histology staining, immunohistochemistry (ICC) and transmission electron microscope (TEM) examinations. Results: After 8-day culture in bioreactor, flow-cytometric analysis confirmed that two cell populations, CD34+CD44+ (HSCs) and Stro-1+CD44+ (MSCs), increased 8-fold and 29-fold respectively, when compared to the values of the MNCs prior to bioreactor treatment. Cell counting revealed that the total cell expansion over 8 days was 9-fold above the number of the input MNCs. Western blotting data confirmed that bioreactor-expanded MSCs population remained in their early-stage with the expression of primitive MSCs markers such as CD105 (endoglin, SH-2) and Vimentin, whereas no expression of differentiation markers including osteocalcin (osteogenesis), Type II collegen (chondrogenesis) and C/EBPα (adipogenesis). Upon differentiation induction, the bioreactor-expanded MSCs were capable of differentiating into osteocytes, chondrocytes, and adipocytes as evidenced by histology staining, ICC and TEM examinations. Discussion: Our study has shown that the percentage of MSCs (Stro-1+CD44+) increased 29 folds in the bone marrow derived MNCs after they have been cultured with Myelocult¢â medium in bioreactor for 8 days. The suspension culture system did not affect the subsequent in vitro proliferation and differentiation potentials of MSCs. Current study indicates that rotary bioreactor may be used to rapidly expand the numbers of traditionally attachment-dependent MSCs from bone marrow-derived MNCs, which may be very useful in clinical tissue engineering applications

Background. Recent advances in materials and manufacturing processes for arthroplasty have allowed fabrication of intricate implant surfaces to facilitate bony attachment. However, refinement and evaluation of these new design strategies is hindered by the cost and complications of animal studies, particularly during early iterations in development process. To address this problem, we have constructed and validated an ex-vivo bone bioreactor culture system to enable empirical testing of candidate structures and materials. In this study, we investigated mineralization of a titanium wire mesh scaffold under both static and dynamic culturing using our ex vivo bioreactor system. Methods. Cancellous cylindrical bone cores were harvested from bovine metatarsals and divided into five groups under different conditions. After incubation for 4 & 7 weeks, the viability of each bone sample was evaluated using Live-Dead assay and microscopic anatomy of cells were determined using histology stain H&E. Matrix deposits on the scaffolds were examined with scanning electron microscopy (SEM) while its chemical composition was measured using energy-dispersive x–ray spectroscopy (EDX). Results. The viability of bone cores was maintained after seven weeks using our protocol and ex vivo system. From SEM images, we found more organic matrix deposition along with crystallite like structures on the metal samples pulled from the bioreactor indicating the initial stages of mineralization. EDX results further confirmed the presence of carbon and calcium phosphates in the matrix. Conclusion. A bone bioreactor can be used a tool alternate to in-vivo for bone ingrowth studies on new implant surfaces or coatings

Introduction: The treatment of bone defects that occurs following fractures, the excision of bone tumours and at revision arthroplasty surgery, often involves the use of either autologous or allogenous bone grafts. However, both grafts have limitations. The aim of tissue engineering is to produce cells within an extracellular matrix that resembles tissue, which can be implanted into a patient to heal a tissue defect. The potential to engineer bone tissue grafts from patients’ autologous cells would improve the treatment of bone defects. Bone marrow contains cells, known as mesenchymal stem cells (MSCs), which have the ability to differentiate into osteoblasts. To create a 3-dimensional structure necessary for the reconstruction of tissue, cells need to be grown on a scaffold, for which hydroxyapatite (HA) was used, as it is osteoconductive. In living bone, increased extravascular perfusion increases new bone formation. Thus, these physiological conditions were reproduced in our novel bioreactor by perfusing MSCs seeded on porous HA scaffolds at a rate of 6ml/hr. Hypotheses: 1. Culture in this bioreactor improves cell penetration through a HA scaffold. 2. MSCs cultured on HA in this bioreactor differentiated into osteoblasts. Method: MSCs were isolated from 8 bone marrow aspirates, which were taken from patients during orthopaedic procedures following informed consent. For each experiment, MSCs from each patient were seeded onto 2 x 1cm. 3. scaffolds. To test cell penetration, the HA scaffolds were cultured for 7 days, then sectioned longitudinally and the number of cells were counted at increasing depths. Observations of MSCs on HA were compared under scanning (SEM) and transmission (TEM) electron microscopy. The HA scaffolds were cultured with MSCs in the bioreactor for 5, 10 & 15 days, after which time alkaline phosphatase (ALP) and type I pro-collagen protein levels were measured. Results: Penetration of cells through the porous HA scaffold was significantly greater when the cells had been cultured in the bioreactor (P< 0.05). Observing MSCs after 7 days in bioreactor culture under SEM, adherent fibroblastic cells formed a network over the HA. However, by 14 days the HA was covered with cuboidal cells, consistent with osteoblasts. TEM results showed that MSCs cultured on HA in the bioreactor produced organised collagen matrix after 28 days. Osteoblastic protein levels were significantly greater at each time point when MSCs were cultured in bioreactor conditions: ALP (P< 0.005) and type I pro-collagen (P< 0.05). Discussion and Conclusions: These results show that when cultured in our novel bioreactor, MSCs penetrated uniformly through the porous HA scaffold, whereas few cells penetrated in static culture conditions. Thus, our bio-reactor significantly improves the 3-dimensional growth of cells, resembling tissue. Moreover, in this study MSCs grown on HA in the bioreactor produced significantly larger amounts of ALP and type I pro-collagen, indicating that the MSCs differentiated into osteoblasts. Observations under TEM showed extracellular collagen matrix production which, when mineralized, produces bone. Therefore, this culture method could potentially be used to convert MSCs, isolated from patients’ bone marrow, into tissue-engineered bone

Bioreactors used in tissue engineering are mostly batch-fed with media added and removed periodically. Continuous flow bioreactors help increase ECM accumulation and cell proliferation, due to continuous flow of fresh media, thus, maintaining a steady extracellular nutrient environment. In previous work, we found chondrocytes cultured in continuous flow bioreactors with 20mM HEPES, accumulated considerably more matrix than static cultures. Hence, the objective of this study is to determine if NaHCO3 helps maintain a more physiological extracellular pH in the bioreactor, thus, enhancing ECM accumulation. Cartilaginous tissue constructs were generated from isolated chondrocytes harvested from the metacarpal joints of 12-18 month old calves. Cells were seeded in high-density 3D cultures (2 million cells/construct). Constructs were cultivated in a continuous flow bioreactor, with and without 14 mM NaHCO3 supplemented media, for 5 weeks, at 37 degrees Celsius, 95% relative humidity and 5% CO2. After 5 weeks of culture the tissue weight, thickness, pH and ECM deposition were determined. From the results obtained (Table 1), it is evident that chondrocytes cultured in the continuous flow bioreactor with 14mM NaHCO3 and 20mM HEPES, proliferated more extensively and produced more ECM than chondrocytes cultured in only 20mM HEPES. Additionally, the NaHCO3 constructs accumulated ECM in both the vertical (thickness) and horizontal (outgrowth) planes. The question then arises, are the effects mediated by improved buffering, or by addition of NaHCO3 itself. There was a significant difference between the pH of media with (pH 7.41) and without NaHCO3 (pH 6.95) supplementation, with no exposure to cells or tissue; when allowed to equilibrate with 5% CO2 at 37 degrees Celsius. However, there was little difference between the media after exposure to cells; after five weeks of culture in the bioreactor (Table 1). Thus, in the bioreactor with bicarbonate present, because of increased cell number and activity, the pH fell 0.54 pH units during the 7 hour residence time in comparison to the bioreactor with no bicarbonate supplementation. With no NaHCO3 supplementation, the extracellular pH of the medium fed to the cells was never above pH 7.0 (Table 1); low pH could account, at least in part, for lower ECM and cell numbers

Short Summary. The present study demonstrated the feasibility of culturing a large number of standardised granular MSC-containing constructs in a packed bed/column bioreactor that can produce sheep MSC-containing constructs to repair critical-size bone defects in sheep model. Introduction. Endogenous tissue regeneration mechanisms do not suffice to repair large segmental long-bone defects. Although autologous bone graft remains the gold standard for bone repair, the pertinent surgical technique is limited. Tissue constructs composed of MSCs seeded onto biocompatible scaffolds have been proposed for repairing bone defects and have been established in clinically-relevant animal models. Producing tissue constructs for healing bone defects of clinically-relevant volume requires a large number of cells to heal an approximately 3 cm segmental bone defect. For this reason, a major challenge is to expand cells from a bone marrow aspirate to a much larger, and sufficient, number of MSCs. In this respect, bioreactor systems which provide a reproducible and well-controlled three-dimensional (3D) environment suitable for either production of multiple or large size tissue constructs are attractive approaches to expand MSCs and obtain MSC-containing constructs of clinical grade. In these bioreactor systems, MSCs loaded onto scaffolds are exposed to fluid flow, a condition that provides both enhanced access to oxygen and nutrients as well as fluid-flow-driven mechanical stimulation to cells. The present study was to evaluate bioreactor containing autologous MSCs loaded on coral scaffolds to repair critical-size bone defects in sheep model. Materials and Methods. Animals: 12 two-year-old, female Pre-Alpes sheep were used and reared in accordance with the European Committee for Care. Three-dimensional, porous scaffolds (each 3×3×3 mm) of natural coral exoskeleton were used as substrates for cell attachment. The packed bed/column bioreactor set-up used in the present study was composed of a vertical column filled with MSC-containing constructs. Sheep MSCs were isolated from sheep bone marrow. MSCs were seeded on scaffolds and cultured overnight under standard cell-culture condition. MSC-containing constructs were r placed into the perfusion bioreactor and were either exposed to a perfusion medium flow rate of 10 mL/min for 7 continuous days. Osteoperiosteal segmental (25 mm) defects were made in the left metatarsal bone of 12 sheep. The defect was either filled with coral scaffolds alone (Group 1; five sheep); or filled with coral scaffolds loaded with MSCs (Group 2; five sheep); or filled with autologous bone graft (Group 3; 2 sheep). Results. At 6 month after implantation, radiographs showed resorption of the coral scaffold in all animals but this process was not complete and not the same in all animal. At 6 month radiographs showed more bone formation in group 2 than in group 1. New bone formation volume in each defect was assessed by micro-computed tomography. Volume of bone healing was higher in group 2 than group 1. Discussion. The potential of MSC-containing constructs in a bioreactor for repairing long segmental critical-sized bone defects in sheep was investigated. In one animal of the group 2 the volume of new bone formation was 2066 mm3 and was similar to the bone volume of group 3 (2300 mm3). Our results may have important implications in bone tissue engineering. We observed that the bone tissue regenerationosteogenic ability of bone constructs processed in bioreactor approached the bone autografts

Introduction: Due to a lack of techniques there is only some data of testing mechanical influence on chondroc-tyes grown in 3-D tissue-culture over several months. The authors developed therfore a new perfusion-chamber to study these mechanical factors in in-vitro tissue culture. Methods: A pneumatic computer-controlled bioreactor which allows the application of pressure between 0,1 – 2 MPa in frequencies less than 1 Hz over a period of several months was developed. The reactor is made of stainless steel and closed to the surrounding atmosphere with a steril, flexible and pressure resistant layer of film. 8 scaffolds with a maximal diameter of 10 mm and a maximal height of 15 mm can be studied independently in respect to pressure parameters. A continous flow of medium allows an excellent nutrition, temperature is controlled by a closed heat unit. In a first system three bioreactors are used simultaneousely. Results: In first cultures of tissues over a period of three months sterility was well contained without any mechanical problems. With a high degree of automation a nearly uncontrolled running was possible

Using the trabecular bone bioreactor (ZETOS) developed in our laboratories we have investigated the formation of bone using the fluorescent bone seeking markers calcein and alizarin red. And the association of bone formation with the increase in stiffness with mechanical loading. 10 mm diameter bone cores 5 mm thick were obtained from the distal radius /ulna of cows obtained at the slaughter house. by precision cutting with diamond saws and keyhole cutters (our pattern) in sterile 7–10°C phosphate buffered saline (PBS) and cultured in a variation of DMEM containing fructose HI GEM. Results: Loading the bone 30x 4,000μ per day resulted in an increase of stiffness of 35%, by day 30 while the non loaded controls decreased in stiffness. Calcein was added at day 27 to the circulating medium for 4 hours and then fresh medium was circulated. On day 30 alazarin red was circulated through the trabecular bone. The bones were subsequently fixed and embedded in resin and sectioned by classical histological techniques. The difference in distance between the two dyes indicated the amount of bone formation. The mechanically loaded bones showed significant evidence of formation and also significant numbers of active osteoclasts indicating high bone turnover. No evidence of necrosis or cartilage formation was found. Formation in unloaded bones was much reduced and on many areas no active osteoblasts could be observed. This is the first demonstration of bone formation ex vivo after 30 days of culture. We gratefully acknowledge support by the German Arthrose Foundation (DAH) and the AO in Davos, CH. DJ is a recipient of a Fork award from the AO

Background. For bone grafting procedures, the use of autologous bone is considered the gold standard, as it is has a better healing capacity compared to other alternatives as allograft and synthetic bone substitutes. However, as there are several drawbacks related to autografting (infection, nerve- or vascular damage, chronic pain problems, abdominal herniation), there has been a targeted effort to improve the healing capacities of synthetic bone substitutes. Aim. To evaluate the performance of a carbonated osteoionductive hydroxyapatite (CHA) scaffold of clinical relevant size (Ø=15mm, H=50mm) in a sheep model of multi level posterolateral intertransverse lumbar spine fusion after activation with autologous bone marrow nuclear cells (BMNC) in a flow perfusion bioreactor. Method. Two groups were included in the study, autograft (n=6) and CHA scaffold (n=6) CHA. A paired design was used between and within the groups as lumbar posterolateral arthrodesis was performed in sheep on two levels (L2-L3, L5-L6) +/− BMNC, respectively. Before implantation, the CHA scaffold was cultured in a flow perfusion bioreactor system with BMNC for 21 days, and the autograft group was supplemented with isolated BMNC during the procedure. Micro tomography was used to evaluate fusion rate and the microarchitectural properties of the explants after an observation period of four months. Results. In the autograft group, the healing rate was 83.3% irrespective of the presence BMNC, and in the CHA group, 66.7% fused in the presence of BMNC, and 33.3% without. The microarchitectural data suggested the autograft group to be superior to the CHA scaffold regarding mechanical properties, however porosity decreased significantly (p=0.001) in the CHA scaffold group suggesting deposition of mineralized bone matrix. Conclusion. Based on the fusion rate and micro architectural properties, we consider the CHA scaffold fully capable of new bone formation, and that the presence of BMNC has a positive effect on the fusion rate in a challenging model of bone healing

Objectives

Induced membrane technique is a relatively new technique in the reconstruction of large bone defects. It involves the implantation of polymethylmethacrylate (PMMA) cement in the bone defects to induce the formation of membranes after radical debridement and reconstruction of bone defects using an autologous cancellous bone graft in a span of four to eight weeks. The purpose of this study was to explore the clinical outcomes of the induced membrane technique for the treatment of post-traumatic osteomyelitis in 32 patients.

One of the mechanisms which controls bone growth, repair remodeling and absorption is mechanical loading. There exists no long-term in vitro model to study bone cells together with their matrix, nor a model that can apply quantitative mechanical forces of physiological amplitudes and frequencies. The analysis of the mechanical properties of bone (Young’s modulus and visco-elastic moduli) on small pieces of bone is also difficult with present devices. We have built a device that can maintain full viability and physiological response of bone for a period of several weeks and integrates all three functions.

10mm diameter bone cores 5 mm thick were obtained from the trabecular bone of the distal ulna of a 24 months old cow by precision cutting with diamond saws and keyhole cutters (our pattern) in sterile 7–10°C phosphate buffered saline (PBS) and cultured in a variation of DMEM containing fructose HI GEM.

Results: The results of these studies have shown that perfusion of trabecular bone can maintain all cells and maintain bone structure for at least 72 days. In conventional methods for bone organ cultures, small bones, such as rat calvaria, quickly start to resorb bone and degenerate. In our perfusion system we see no evidence of change.. Initial experiments have indicated that there are 2 visco-elastic moduli of bone with different time constants, that the elastic modulus of trabecular bone varies is site dependant and that loading to 0.4% compression raises prostaglandin E2 and insulin-like growth factor 1 within a few hours. Mechanical stiffness of bone is increased by 35% when loaded for 20 days at 4,000μ, and decreases by 25% when not loaded. PTH at 10-10M increases stiffness over the load effect and 10-6M PTH decreases stiffness even in the presence of loading. Active osteoclasts are seen during the whole culture period indicating that the stem cells are present and functional.

We gratefully acknowledge support by the German Arthrose Foundation (DAH) and the AO in Davos, CH.

Introduction: Tissue engineered scaffolds require vascularization to 1) enhance nutrient exchange and 2) provide cells needed to build new tissue. Cell-seeded scaffolds; bioreactors-- require rapid penetration of vessels or enhanced fluid percolation to keep their contents alive until normal nutrient exchange can be established. Bone fluid flow depends on a pumping system which drives percolation through its own matrix. Recent interest in the pumping mechanism has resulted in bone fluid flow models, which link the pumps to bending of bone by muscle contraction and compression-tension cycles from weight-bearing during locomotion. The present authors have proposed that capillary filtration, the source of the percolating fluid, is sufficiently enhanced by soliton pressure waves in blood driven by the muscle pump during exercise to provide a significant hydraulic pressure component to bone fluid percolating through bone and any bone-implanted scaffold. A proposal and some preliminary results from a pilot project suggesting enhancement of capillary filtration by the muscle pump is presented.

Materials and Methods: Optical bone chambers were implanted in adult New Zealand White female rabbits. Chamber construction and implantation were as usual1. At the third week post-op, chamber ends were exposed and weekly intravital microscopy commenced. Transcutaneous electrical stimulation was administered with a ToneATronic® TENS at 85V, 80mA and 2Hz. The stimulator was applied externally over the gastrocnemius muscle. A fluorescence digital image was obtained before 30 minutes of application of transcutaneous electrical nerve stimulation (TENS) after injection of FITC-D70. Blood samples were obtained from an aural vein in the ear opposite that being injected with the fluorescent dye after each injection. Blood concentration of dye was determined with a SPEX Fluoromax-3 spectrofluorometer for both serum (absolute concentration) and whole blood (to detect differences which would make fluorescence in vessels an inaccurate indicator of red blood cell color contamination). For analysis, four vessels were chosen and the average dye concentration profiles before and after 30 minutes of stimulation were obtained.

Results: Results are shown in Figure 1. Extravasated dye levels in TENS rabbits were markedly higher than those in controls. Analysis of profiles using an erfc-based diffusion-convection discrimination model2 showed that extravasation was convective.

Discussion: These data are consistent with significant contribution to convective percolation of bone fluid through implanted scaffolds by muscle pump-driven extravasating fluid. They do not, however, answer two critical questions: 1) Is the magnitude of this convection a major component of flow through the scaffold? 2) What are the relative contributions of skeletal muscle-generated intravascular pressure solitons and incompressible fluid transmission of bone bending pressure to the convective flow observed? Additional studies with released gastrocnemius muscles are in progress.