Fabrication of individual scaffolds based on a patient-specific alveolar bone defect model

Fabricating individualized tissue engineering scaffolds based on the three-dimensional shape of patient bone defects is required for the successful clinical application of bone tissue engineering. However, there are currently no reported studies of individualized bone tissue engineering scaffolds that truly reproduce a patient-specific bone defect. We fabricated individualized tissue engineering scaffolds based on alveolar bone defects. The individualized poly(lactide-co-glycolide) and tricalcium phosphate composite scaffolds were custom-made by acquiring the three-dimensional model through computed tomography, which was input into the computer-aided low-temperature deposition manufacturing system. The three-dimensional shape of the fabricated scaffold was identical to the patient-specific alveolar bone defects, with an average macropore diameter of 380 μm, micropore diameters ranging from 3 to 5 μm, and an average porosity of 87.4%. The mechanical properties of the scaffold were similar to adult cancellous bone. Scaffold biocompatibility was confirmed by attachment and proliferation of human bone marrow mesenchymal stem cells. Successful realization of individualized scaffold fabrication will enable clinical application of tissue-engineered bone at an early date.     

Protein- and gene-based tissue engineering in bone repair

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Ex vivo gene therapy in autologous critical-size craniofacial bone regeneration

In therapeutic bone repairs, autologous bone grafts, conventional or vascularized allografts, and biocompatible artificial bone substitutes all have their shortcomings. The bone formed from peptides [recombinant human bone morphogenetic proteins (BMPs)], demineralized bone powder, or a combination of both is small in size. Tissue engineering may be an alternative for cranial bone repair. In this study, the authors developed an animal model to test the hypothesis that replication-defective, adenovirus-mediated human BMP-2 gene transfer to bone marrow stromal cells enhances the autologous bone formation for repairing a critical-size craniofacial defect. The mesenchymal stromal cells of miniature swine were separated from the iliac crest aspirate and expanded in monolayer culture 1 month before implantation. The cultured mesenchymal stromal cells were infected with recombinant, replication-defective human adenovirus BMP-2, 7 days before implantation. Bilateral 2 x 5-cm2 cranial defects were created, leaving no osteogenic periosteum and dura behind. Mesenchymal stromal cells at 5 x 10(7)/ml were mixed with collagen type I to form mesenchymal stromal cell/polymer constructs. Mesenchymal stromal cells used for the control site were infected with adenovirus beta-Gal under the same conditions. After 6 weeks and 3 months, 10 miniature swine were euthanized and the cranium repair was examined. Near-complete repair of the critical-size cranial defect by tissue-engineered mesenchymal stromal cell/collagen type I construct was observed. The new bone formation area (in square centimeters) measured by three-dimensional computed tomography demonstrated that the improvement from 6 weeks to 3 months was significantly greater on the experimental side than on the control side (2.15 cm2 versus 0.54 cm2, p < 0.001) and significantly greater at 3 months than at 6 weeks (2.13 cm2 versus 0.52 cm2, p < 0.001). The difference between the experimental and control groups was significant at 3 months (mean difference, 2.13 cm2; p < 0.001). The maximal compressive strength of the new bone was similar to that of the normal cranial bone when evaluated by biomechanical testing (cranium bone versus tissue-engineered bone, 88.646 +/- 5.121 MPa versus 80.536 +/- 19.302 MPa; p = 0.227). Adenovirus was absent from all constructs by immunochemical staining at 6 weeks and 3 months after implantation. The successful repair of cranial defects in this experiment demonstrates the efficacy of the integration of the autologous stem cell concept, gene medicine, and polymers in producing tissue-engineered bone.     

Scaffold-based bone engineering by using genetically modified cells

The first generation of clinically applied tissue engineering concepts in the area of skin, cartilage and bone marrow regeneration was based on the isolation, expansion and implantation of cells from the patient's own tissue. Although successful in selective treatments, tissue engineering needs to overcome major challenges to allow widespread clinical application with predictable outcomes. One challenge is to present the cells in a matrix to the implantation site to allow the cells to survive the wound healing contraction forces, tissue remodeling in certain tissues such as bone and biomechanical loading. Hence, several tissue engineering strategies focus on the development of load-bearing scaffold/cell constructs. From a cell source point of view, bone engineers face challenges to isolate and expand cells with the highest potential to form osseous tissue along with harvesting tissue without extensive donor site morbidity. A major hurdle to tissue engineering is de-differentiation and limited ability to control cell phenotype following in vitro expansion. Due to early successes with genetic engineering, bone tissue engineers have used different strategies to genetically alter various types of mesenchymal cells to enhance the mineralization capacity of tissue-engineered scaffold/cell constructs. Although the development of multi-component scaffold/osteogenic cell constructs requires a combination of interdisciplinary research strategies, the following review is limited to describe the general aspects of bone engineering and to present overall directions of technology platforms, which include a genetic engineering component. This paper reviews the most recent work in the field and discusses the concepts developed and executed by a collaborative effort of the multi-disciplinary teams of the two authors.     

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.     

Evaluation of bone marrow derived mesenchymal stem cells for full-thickness wound healing in comparison to tissue engineered chitosan scaffold in rabbit

Background

Chronic wounds present a major challenge in modern medicine. Even under optimal conditions, the healing process may lead to scarring and fibrosis. The ability of mesenchymal stem cells (MSCs) to differentiate into other cell types makes these cells an attractive therapeutic tool for cell transplantation. Both tissue-engineered construct and MSC therapy are among the current wound healing procedures and potential care. Chitosan has been widely applied in tissue engineering because of its biocompatibility and biodegradability.

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Biological and molecular profile of fracture non‐union tissue: current insights

Delayed bone healing and non‐union occur in approximately 10% of long bone fractures. Despite intense investigations and progress in understanding the processes governing bone healing, the specific pathophysiological characteristics of the local microenvironment leading to non‐union remain obscure. The clinical findings and radiographic features remain the two important landmarks of diagnosing non‐unions and even when the diagnosis is established there is debate on the ideal timing and mode of intervention. In an attempt to understand better the pathophysiological processes involved in the development of fracture non‐union, a number of studies have endeavoured to investigate the biological profile of tissue obtained from the non‐union site and analyse any differences or similarities of tissue obtained from different types of non‐unions. In the herein study, we present the existing evidence of the biological and molecular profile of fracture non‐union tissue.     

Bone Marrow Aspiration Concentrate and Platelet Rich Plasma for Osteochondral Repair in a Porcine Osteochondral Defect Model

Background

Bone marrow aspiration concentrate (BMAC) may possess a high potency for cartilage and osseous defect healing because it contains stem cells and multiple growth factors. Alternatively, platelet rich plasma (PRP), which contains a cocktail of multiple growth factors released from enriched activated thrombocytes may potentially stimulate the mesenchymal stem cells (MSCs) in bone marrow to proliferate and differentiate.

Methods

A critical size osteochondral defect (10×6 mm) in both medial femoral condyles was created in 14 Goettinger mini-pigs. All animals were randomized into the following four groups: biphasic scaffold alone (TRUFIT BGS, Smith & Nephew, USA), scaffold with PRP, scaffold with BMAC and scaffold in combination with BMAC and PRP. After 26 weeks all animals were euthanized and histological slides were cut, stained and evaluated using a histological score and immunohistochemistry.

Results

The thrombocyte number was significantly increased (p = 0.049) in PRP compared to whole blood. In addition the concentration of the measured growth factors in PRP such as BMP-2, BMP-7, VEGF, TGF-β1 and PDGF were significantly increased when compared to whole blood (p<0.05). In the defects of the therapy groups areas of chondrogenic tissue were present, which stained blue with toluidine blue and positively for collagen type II. Adding BMAC or PRP in a biphasic scaffold led to a significant improvement of the histological score compared to the control group, but the combination of BMAC and PRP did not further enhance the histological score.

Conclusions

The clinical application of BMAC or PRP in osteochondral defect healing is attractive because of their autologous origin and cost-effectiveness. Adding either PRP or BMAC to a biphasic scaffold led to a significantly better healing of osteochondral defects compared with the control group. However, the combination of both therapies did not further enhance healing.     

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

Evaluation of new porous β-tri-calcium phosphate ceramic as bone substitute in goat model

The present study was carried out to evaluate the porous β-tri-calcium phosphate (TCP) (prepared by aqueous solution combustion technique) as bone substitute and compared with normal healing in 12 adult Black Bengal goats on the basis of clinical and radiographic findings, histological studies, oxytetracycline labeling, angiography studies (on day 90). Bone defects created in the diaphysis of radius were left unfilled in control animals (group I); while in treated (group II) animals the defects were filled with porous TCP blocks. The three months study showed no marked acute inflammatory reactions in all animals, wound healing was uneventful and the implants were clinically stable in the bone. Radiological studies showed presence of unabsorbed implants which acted as a scaffold for new bone growth across the defect whereas in control animals the defect was more or less same except that the newly formed bony tissue was less organized. Histological section showed moderately differentiated lamellar bone in the cortical part with presence of woven bone at peripheral cortex whereas control animals showed moderate fibro-collagenisation and good amount of marrow material, fat cells and blood vessels. Oxytetracycline labeling study showed crossing over of new bony trabeculae along with presence of resorption cavities within the new osteoid tissues whereas in group I, the process of new bone formation was active from both the ends; the defect site appeared as a homogenous non-fluorescent area. Angiogram of the animals in control showed uniform angiogenesis in the defect site with establishment of trans transplant angiogenesis, whereas in group II there was complete trans transplant shunting of blood vessels communication. The results of this study pointed out that the porous TCP promoted extensive bone formation over the entire extension of the defect in comparison to control group, thus conforming their biological osteoconductive property.     

Studies on antigenic competition. Efforts to identify the cellular basis of competition using a cell transfer system

Antigenic competition was studied in a cell transfer system. The effect of reconstituting lethally irradiated mice with various numbers of thymus or bone marrow cells on the extent of antigenic competition was evaluated. Brucella or burro erythrocytes both caused inhibition of the immune response (Plaque-forming cell) to sheep erythrocytes when given two days prior to the test antigen. Varying the dose of thymus cells, or of bone marrow cells did not alter the degree of competition. Competition was observed even when the competing antigen was injected with bone marrow cells alone two days before the test antigen and thymus cells. The results suggest that mechanisms other than T-cell activation alone must be considered as operative in some models of antigenic competition.     

Repair of rabbit ulna segmental bone defect using freshly isolated adipose-derived stromal vascular fraction

Background aimsStromal vascular fractions (SVF) from adipose tissue have heterogeneous cell populations, and include multipotent adipose-derived stem cells. The advantages of using of SVF include the avoidance of an additional culture period, a reduced risk of extensive cell contamination, and cost-effectiveness.MethodsUnilateral 20-mm mid-diaphyseal segmental defects in rabbit ulna were treated with one of the following: polylactic glycolic acid (PLGA) scaffold alone (group 1, control), a PLGA scaffold with undifferentiated SVF cells (group 2), or a PLGA scaffold with osteogenically differentiated SVF cells (group 3). At 8 weeks after implantation, five rabbits in each treatment group were killed to assess bone defect healing by plain radiography, quantitative microcomputed tomography and histology.ResultsThe SVF cells were well grown on PLGA scaffolds and expressed type I collagen and alkaline phosphatase (ALP). The intensity of ALP and OPN gene expressions in osteogenic medium culture were increased from 14 days to 28 days. In vivo evaluations at 8 weeks showed that treatment of SVF cells with or without osteogenic differentiation resulted in more bone formation in the critically sized segmental defects than PLGA scaffold alone. Osteogenically differentiated SVF cells significantly enhanced bone healing compared with undifferentiated SVF cells.ConclusionsAdipose-derived stromal SVF showed osteogenic potential in vitro. Accordingly, SVF could provide a cell source for bone tissue engineering. However, treatment with uncultured SVF cells on bone healing was not satisfactory in the in vivo animal model.     

B lymphocyte precursors in human bone marrow: an analysis of normal individuals and patients with antibody-deficiency states.

Lymphoid cells containing cytoplasmic IgM but lacking stable surface IgM are believed to be the direct precursors of B lymphocytes. We have characterized these pre-B cells in the bone marrow of normal individuals and patients with a variety of immunoglobulin deficiencies or hematologic disorders by using immunofluorescence and autoradiography. Pre-B cells comprised 5.8 +/- 5.7% of lymphoid cells in normal bone marrow. Eleven patients with infantile X-linked agammaglobulinemia (X-LA) lacked B lymphocytes but had a normal frequency (3.8 +/- 3.6%) of bone marrow pre-B cells. A smaller proportion of marrow pre-B cells from patients with X-LA were engaged in spontaneous DNA synthesis than was found for normal controls. In individuals other than the group with X-LA, the number of circulating B cells was positively correlated with the frequency of marrow pre-B cells. These results indicate that patients with X-LA have a defect in maturation of pre-B cells, and suggest that some patients with acquired B lymphocyte deficiency may have lost the capacity to generate pre-B cells from stem cells.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

T cell regulation of interferon-alpha/beta (IFN-alpha/beta) production by alloantigen-stimulated bone marrow cells

We have previously reported that mouse bone marrow cells produce high levels of interferon-alpha/beta (IFN-alpha/beta) after 5 to 6 days of in vitro culture with irradiated allogenic spleen cells. The current study was initiated to determine whether or not T cells are important for alloantigen-induced IFN-alpha/beta production by mouse bone marrow cells. Bone marrow cells and spleen cells were obtained from C57BL/6 mice. These cells were treated with different monoclonal antisera and complement, and then were cultured 5 to 6 days with irradiated DBA spleen cells. The results from these experiments indicated that optimal IFN-alpha/beta production by alloantigen-stimulated bone marrow cells required Lyt-1+2+ T cells. In addition, when bone marrow cells obtained from nu/nu B10 mice were cultured with alloantigen, only low levels of IFN were produced when compared with IFN production by bone marrow cells obtained from normal littermate B10 mice. The addition of nylon wool-enriched splenic T cells to cultures containing bone marrow cells and alloantigen resulted in an augmentation of IFN-alpha/beta production by three-fold to fivefold. Furthermore, bone marrow cells obtained from alloantigen-immunized mice produced much higher levels of IFN-alpha/beta and in a shorter period of time (2 to 3 days) when compared with bone marrow cells obtained from control or non-immunized mice. Cyclosporin A (CsA) has been shown to inhibit predominantly T cell-dependent responses. The effect of CsA on IFN production by alloantigen-stimulated bone marrow and spleen cells was investigated. The addition of CsA at concentrations as low as 0.1 micrograms/ml inhibited not only IFN-gamma production by alloantigen-stimulated spleen cells, but also IFN-alpha/beta production by alloantigen-stimulated bone marrow cells. In contrast, IFN-alpha/beta production by Newcastle disease virus-infected spleen cells, bone marrow cells, or L cells was not inhibited by the addition of CsA (1 microgram/ml). Thus, the ability of bone marrow cells to produce high levels of IFN-alpha/beta after in vitro culture with alloantigen is dependent upon T cells resident in the bone marrow. IFN-alpha/beta production by alloantigen-stimulated bone marrow cells may play a major role in the pathogenesis associated with graft-vs-host disease and in T cell regulation of hematopoiesis.     

Effects of Cell-Attachment and Extracellular Matrix on Bone Formation In Vivo in Collagen-Hydroxyapatite Scaffolds

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.