The influence of polyimide MP-1™ wear particles on a rodent closed fracture healing model.

Joint replacements provide pain free movement for the injured or our aging population. Current prothesis mainly consist of hard metal on metal, or ceramic femoral head on ultra-high-molecular weight polyethylene (UHMWPE). In this study, a rodent fracture model was used to test the influence of wear debris from a high-performance polymer (polyimide MP-1™). Saline, MP-1™ Low Dose in Saline (1%), or MP-1 High Dose (2%) in Saline was injected directly into a standard closed unilateral femoral fracture in 12-week old Sprague Dawley rats (n = 25) for 1, 3 and 6 weeks. Endpoints included radiography, micro-computed tomography, mechanical testing and paraffin histology. No adverse effects from the wear particles were observed from the current study based on radiology, mechanical or histological data. Although the particles were present, histological analysis revealed a progression in healing between the Polyimide treated groups and the non-treated saline control groups over the duration of 1, 3, and 6 weeks, with no inhibition from the particles. The MP-1™ wear debris generated are larger than 1 µm thus are not able to be engulfed by macrophages and cause osteolysis. This family of polymers (polyimides) may be an ideal material to consider for articulating joints and other implants in the human body.

Lateral fenestration of lumbar intervertebral discs in rabbits: development and characterisation of an in vivo preclinical model with multi-modal endpoint analysis.

PURPOSE: To evaluate the biological and biomechanical effects of fenestration/microdiscectomy in an in vivo rabbit model, and in doing so, create a preclinical animal model of IVDD. METHODS: Lateral lumbar IVD fenestration was performed in vivo as single- (L3/4; n = 12) and multi-level (L2/3, L3/4, L4/5; n = 12) fenestration in skeletally mature 6-month-old New Zealand White rabbits. Radiographic, micro-CT, micro-MRI, non-destructive robotic range of motion, and histological evaluations were performed 6- and 12-weeks postoperatively. Independent t tests, one-way and two-way ANOVA and Kruskal-Wallis tests were used for parametric and nonparametric data, respectively. Statistical significance was set at P < 0.05. RESULTS: All rabbits recovered uneventfully from surgery and ambulated normally. Radiographs and micro-CT demonstrated marked reactive proliferative osseous changes and endplate sclerosis at fenestrated IVDs. Range of motion at the fenestrated disc space was significantly reduced compared to intact controls at 6- and 12-weeks postoperatively (P < 0.05). Mean disc height index percentage for fenestrated IVDs was significantly lower than adjacent, non-operated IVDs for both single and multi-level groups, at 6 and 12 weeks (P < 0.001). Pfirrmann MRI IVDD and histological grading scores were significantly higher for fenestrated IVDs compared to non-operated adjacent and age-matched control IVDs for single and multi-level groups at 6 and 12 weeks (P < 0.001). CONCLUSIONS: Fenestration, akin to microdiscectomy, demonstrated significant biological, and biomechanical effects in this in vivo rabbit model and warrants consideration by veterinary and human spine surgeons. This described model may be suitable for preclinical in vivo evaluation of therapeutic strategies for IVDD in veterinary and human patients.

Mesoderm-derived PDGFRA+ cells regulate the emergence of hematopoietic stem cells in the dorsal aorta.

Mouse haematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5), on the ventral surface of the dorsal aorta, by endothelial-to-haematopoietic transition. We investigated whether mesenchymal stem cells, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta-gonad-mesonephros and contribute to the development of the dorsal aorta and endothelial-to-haematopoietic transition. Here we show that mesoderm-derived PDGFRA+ stromal cells (Mesp1der PSCs) contribute to the haemogenic endothelium of the dorsal aorta and populate the E10.5-E11.5 aorta-gonad-mesonephros but by E13.5 were replaced by neural-crest-derived PSCs (Wnt1der PSCs). Co-aggregating non-haemogenic endothelial cells with Mesp1der PSCs but not Wnt1der PSCs resulted in activation of a haematopoietic transcriptional programme in endothelial cells and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA or BMP, WNT and NOTCH signalling interrupted this reprogramming event. Together, aorta-gonad-mesonephros Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

Physico-Chemical Characteristics and Posterolateral Fusion Performance of Biphasic Calcium Phosphate with Submicron Needle-Shaped Surface Topography Combined with a Novel Polymer Binder.

A biphasic calcium phosphate with submicron needle-shaped surface topography combined with a novel polyethylene glycol/polylactic acid triblock copolymer binder (BCP-EP) was investigated in this study. This study aims to evaluate the composition, degradation mechanism and bioactivity of BCP-EP in vitro, and its in vivo performance as an autograft bone graft (ABG) extender in a rabbit Posterolateral Fusion (PLF) model. The characterization of BCP-EP and its in vitro degradation products showed that the binder hydrolyses rapidly into lactic acid, lactide oligomers and unaltered PEG (polyethylene glycol) without altering the BCP granules and their characteristic submicron needle-shaped surface topography. The bioactivity of BCP-EP after immersion in SBF revealed a progressive surface mineralization. In vivo, BCP-EP was assessed in a rabbit PLF model by radiography, manual palpation, histology and histomorphometry up to 12 weeks post-implantation. Twenty skeletally mature New Zealand (NZ) White Rabbits underwent single-level intertransverse process PLF surgery at L4/5 using (1) autologous bone graft (ABG) alone or (2) by mixing in a 1:1 ratio with BCP-EP (BCP-EP/ABG). After 3 days of implantation, histology showed the BCP granules were in direct contact with tissues and cells. After 12 weeks, material resorption and mature bone formation were observed, which resulted in solid fusion between the two transverse processes, following all assessment methods. BCP-EP/ABG showed comparable fusion rates with ABG at 12 weeks, and no graft migration or adverse reaction were noted at the implantation site nor in distant organs.

Temporal response of an injectable calcium phosphate material in a critical size defect.

BACKGROUND: Calcium phosphate-based bone graft substitutes are used to facilitate healing in bony defects caused by trauma or created during surgery. Here, we present an injectable calcium phosphate-based bone void filler that has been purposefully formulated with hyaluronic acid to offer a longer working time for ease of injection into bony defects that are difficult to access during minimally invasive surgery. METHODS: The bone substitute material deliverability and physical properties were characterized, and in vivo response was evaluated in a critical size distal femur defect in skeletally mature rabbits to 26 weeks. The interface with the host bone, implant degradation, and resorption were assessed with time. RESULTS: The calcium phosphate bone substitute material could be injected as a paste within the working time window of 7-18 min, and then self-cured at body temperature within 10 min. The material reached a maximum ultimate compressive strength of 8.20 ± 0.95 MPa, similar to trabecular bone. The material was found to be biocompatible and osteoconductive in vivo out to 26 weeks, with new bone formation and normal bone architecture observed at 6 weeks, as demonstrated by histological evaluation, microcomputed tomography, and radiographic evaluation. CONCLUSIONS: These findings show that the material properties and performance are well suited for minimally invasive percutaneous delivery applications.

The efficacy of a nanosynthetic bone graft substitute as a bone graft extender in rabbit posterolateral fusion.

BACKGROUND CONTEXT: Synthetic bone graft substitutes are commonly used in spinal fusion surgery. Preclinical data in a model of spinal fusion to support their efficacy is an important component in clinical adoption to understand how these materials provide a biological and mechanical role in spinal fusion. PURPOSE: To evaluate the in vivo response of a nanosynthetic silicated calcium phosphate putty (OstP) combined with autograft compared to autograft alone or a collagen-biphasic calcium phosphate putty (MasP) combined with autograft in a rabbit spinal fusion model. STUDY DESIGN: Efficacy of a nanosynthetic silicated calcium phosphate putty as an extender to autograft was studied in an experimental animal model of posterolateral spinal fusion at 6, 9, 12 and 26 weeks, compared to a predicate device. METHODS: Skeletally mature female New Zealand White rabbits (70) underwent single level bilateral posterolateral intertransverse process lumbar fusion, using either autograft alone (AG), a nanosynthetic silicated calcium phosphate putty (OstP) combined with autograft (1:1), or a collagen-biphasic calcium phosphate putty (MasP) combined with autograft (1:1). Iliac crest autograft was harvested for each group, and a total of 2 cc of graft material was implanted in the posterolateral gutters per side. Fusion success was assessed at all time points by manual palpation, radiographic assessment, micro-CT and at 12 weeks only using non-destructive range of motion testing. Tissue response, bone formation and graft resorption were assessed by decalcified paraffin histology and by histomorphometry of PMMA embedded sections. RESULTS: Assessment of fusion by manual palpation at the 12 week endpoint showed 7 out of 8 (87.5%) bilateral fusions in the OstP extender group, 4 out of 8 (50%) fusions in the MasP extender group, and 6 out of 8 (75%) fusions in the autograft alone group. Similar trends were observed with fusion scores of radiographic and micro-CT data. Histology showed a normal healing response in all groups, and increased bone formation in the OstP extender group at all timepoints compared to the MasP extender group. New bone formed directly on the OstP granule surface within the fusion mass while this was not a feature of the Collagen-Biphasic CaP material. After 26 weeks the OstP extender group exhibited 100% fusions (5 out of 5) by all measures, whereas the MasP extender group resulted in bilateral fusions in 3 out of 5 (60%), assessed by manual palpation, and fusion of only 20 and 0% by radiograph and micro-CT scoring, respectively. Histology at 26 weeks showed consistent bridging of bone between the transverse processes in the Ost P extender group, but this was not observed in the MasP extender group. CONCLUSIONS: The nanosynthetic bone graft substituted studied here, used as an extender to autograft, showed a progression to fusion between 6 and 12 weeks that was similar to that observed with autograft alone, and showed excellent fusion outcomes, bone formation and graft resorption at 26 weeks. CLINICAL SIGNIFICANCE: This preclinical study showed that the novel nanosynthetic silicated CaP putty, when combined with autograft, achieved equivalent fusion outcomes to autograft. The development of synthetic bone grafts that demonstrate efficacy in such models can eliminate the need for excessive autograft harvest and results from this preclinical study supports their effective use in spinal fusion surgery.

Single level posterolateral lumbar fusion in a New Zealand White rabbit (Oryctolagus cuniculus) model: Surgical anatomy, operative technique, autograft fusion rates, and perioperative care.

INTRODUCTION: The posterolateral lumbar fusion (PLF) New Zealand White (NZW) (Oryctolagus cuniculus) rabbit model is a long-standing surgical technique for the preclinical evaluation of materials for spinal fusion. A detailed understanding of lumbar spine anatomy and perioperative care requirements of rabbits is imperative for correct execution of the model both scientifically and ethically. This study describes the preoperative procedures and surgical techniques used in single level PLF in a NZW rabbit model as it pertains to the animal husbandry, lumbar spine anatomy, anesthesia, surgical approach, and perioperative care of rabbits in a research setting. MATERIALS AND METHODS: We describe the surgical technique and perioperative patient care for single level PLF in a NZW rabbit model. Medical records from a single research facility were retrospectively reviewed for adult NZW rabbits that underwent single level PLF (L4-L5) between January 2016 and December 2019. The number of lumbar vertebrae per rabbit, fusion rates at 12 weeks using iliac crest autograft and complications are reported. Skeletal maturity was confirmed by preoperative fluoroscopic and radiographic documented closure of hindlimb physes. RESULTS: The PLF rabbit surgical model and perioperative patient care is described. PLF was performed in 868 adult female entire NZW rabbits. The majority of rabbits had seven lumbar vertebrae (620/868; 71.4%), followed by six (221/868; 25.5%), and eight (27/868; 3.1%). Fusion rates at 12 weeks for PLF using iliac crest autograft as assessed by manual palpation and radiographic assessment was 76.9% and 70.0%, respectively. Postoperative complications included occasional partial autograft site wound dehiscence due to self-trauma. CONCLUSIONS: For PLF rabbit models, a detailed understanding of the surgical technique, rabbit lumbar anatomy including number of lumbar vertebrae, and dietary and husbandry requirements of rabbits, is essential for execution of the model and animal welfare.

Induction of muscle-regenerative multipotent stem cells from human adipocytes by PDGF-AB and 5-azacytidine.

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factor-AB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissue-derived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocyte-derived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.

Moment arm function dictates patella sagittal height anatomy: Rabbit epiphysiodesis model alters limb length ratios and subsequent patellofemoral anatomical development.

Patellofemoral anatomical dysplasia is associated with patellofemoral instability and pain. The closure of the knee physis occurs at the same age as the peak incidence of patellofemoral dislocation. This study determined the effect on the patellofemoral anatomical development in a rabbit epiphysiodesis model. Twenty-four skeletally immature New Zealand White rabbits were divided into three groups (a) distal femur epiphysiodesis (FE) (b) proximal tibia epiphysiodesis (TE) (c) control; no epiphysiodesis (C) performed at 6 weeks of age. The primary endpoint was shape analysis using three-dimensional reconstructions of micro-computed tomographys (CTs) performed at 30 weeks of age. The limb length ratios (femur:tibia) were significantly different for both FE (mean 0.72, SD 0.0381, P < .001) and TE (mean 0.91, SD 0.0383, P < .001) treatment groups compared to control (mean 0.81, SD 0.0073). Patella height, as measured from the most distal point of the patella to the tibial joint surface (modified Caton-Deschamps measurement), was lower (baja) in the FE and higher (alta) for the TE, compared with the control group. Our findings suggest femoral and tibial shortening can influence the development of the patellofemoral joint, which may be dictated by moment arm function and is potentially responsible for the etiology of patella alta. Future studies are warranted to explore this association further with the view for the development of treatment options for patella alta in human patients.

Comparative osteoconductivity of bone void fillers with antibiotics in a critical size bone defect model.

The study aimed to evaluate the comparative osteoconductivity of three commercially available bone void fillers containing gentamicin with respect to new bone, growth, host tissue response and resorption of the implant material. Defects were created in the cancellous bone of the distal femur and proximal tibia of 12-skeletally mature sheep and filled with three commercially available bone void fillers containing gentamicin (Stimulan-G, Cerament-G, Herafill-G). Peripheral blood was taken pre-operatively and at the time of implantation, as well as at intermittent timepoints following surgery to determine systemic gentamicin levels (5-,15- and 30- minutes, 1, 2, 3, 6, 12, 24, 48- and 72-hours, 3-, 6- and 12-weeks). Decalcified, embedded samples were stained with haematoxylin and eosin (H&E) and used to assess the host tissue response and the formation of new bone in the presence of test implant materials. No adverse reactions were noted at harvest at any time points for any cancellous implantation sites with the various implant materials. Comparative microCT analysis of the Stimulan-G, Cerament-G and Herafill-G test materials revealed a similar increase in bone surface area and volume between animals implanted with Stimulan-G or Cerament-G test materials. Animals implanted with Herafill-G test materials demonstrated the lowest increases in bone volume and surface area of the test materials tested, at levels similar to the negative control sites. By 12-weeks, Stimulan-G defects were completely closed with mature bone and bone marrow whilst the Cerament-G material was still present after 12 weeks by histological examination. In conclusion, this study demonstrated differences in the bone regenerative capacity of a range of bone void fillers in an in vivo setting.

Effects of SCCO2, Gamma Irradiation, and Sodium Dodecyl Sulfate Treatments on the Initial Properties of Tendon Allografts.

Sterile and decellularized allograft tendons are viable biomaterials used in reconstructive surgeries for dense connective tissue injuries. Established allograft processing techniques including gamma irradiation and sodium dodecyl sulfate (SDS) can affect tissue integrity. Supercritical carbon dioxide (SCCO2) represents a novel alternative that has the potential to decellularize and sterilize tendons with minimized exposure to denaturants, shortened treatment time, lack of toxic residues, and superior tissue penetration, and thus efficacy. This study attempted to develop a single-step hybrid decellularization and sterilization protocol for tendons that involved SCCO2 treatment with various chemical additives. The processed tendons were evaluated with mechanical testing, histology, scanning electron microscopy (SEM), and Fourier-transform infrared (FTIR) spectroscopy. Uniaxial mechanical testing showed that tendons treated with SCCO2 and additive NovaKillTM Gen2 and 0.1% SDS had significantly higher (p < 0.05) ultimate tensile stress (UTS) and Young's modulus compared to gamma-irradiated and standard-SDS-treated tendons. This was corroborated by the ultrastructural intactness of SCCO2-treated tendons as examined by SEM and FTIR spectroscopy, which was not preserved in gamma-irradiated and standard SDS-treated tendons. However, complete decellularization was not achieved by the experimented SCCO2-SDS protocols used in this study. The present study therefore serves as a concrete starting point for development of an SCCO2-based combined sterilization and decellularization protocol for allograft tendons, where additive choice is to be optimized.

The effect of surgery on patellar tendinopathy: Novel use of MRI questions the exploitability of the rat collagenase model to humans.

BACKGROUND: patellar tendinopathy is an overuse condition most commonly affecting jumping athletes. Surgery is reserved for refractory cases; however, it lacks high level clinical evidence and basic science to support its use. The purpose of this study was to determine the biomechanical and histological response of surgical excision on patellar tendinopathy in the rat collagenase tendinopathy model and correlate MRI findings. METHODS: Forty-eight Long Evans rats were divided into three groups: i) no patellar tendinopathy with surgical excision, ii) patella tendinopathy with surgical excision, and iii) patellar tendinopathy with no surgical excision. Endpoints included histology, mechanical testing, and MRI pre- and post-surgical intervention at one and four weeks. RESULTS: No difference in failure load or histological grading was seen between the groups at all time points. MRIs showed initial loss of tendon continuity followed by complete healing with elongated and thickened tendons in all groups. CONCLUSIONS: While other research has reported immunohistochemistry and histology of collagenase-induced tendinopathy may be correlated with human pathogenesis, the novel MRI findings from our study suggest that the rat collagenase tendinopathy surgical model may be limited when extrapolating to humans. Further work is needed to determine if any correlation exists between the dosing, location, and animal effect of the collagenase injection model with MRI findings. This is needed before any collagenase model can be used to determine the effect of surgery in the pathogenic response to patella tendinopathy.

Suture wear particles cause a significant inflammatory response in a murine synovial airpouch model.

BACKGROUND: Commonly used contemporary orthopaedic sutures have been identified as a potential causative factor in the development of post-arthroscopic glenohumeral chondrolysis. Currently, little is known about the body's immune response to these materials. The aim of this study was to examine the biological response of synovial tissue to three commonly used orthopaedic sutures, using a murine airpouch model. METHODS: Fifty rats were used in this study (ten per group). An airpouch was created in each rat, and test materials were implanted. Test materials consisted of an intact polyethylene terephthalate suture with a polybutilate coating (suture A), an intact polyethylene suture braided around a central polydiaxannone core (suture B), an intact polyethylene/polyester cobraid suture with a silicone coating (suture C), and particles of suture C (particles C). Rats were sacrificed at 1 or 4 weeks following implantation. Histological (multinucleated giant cell count) and immunohistochemical (expression of matrix metalloproteinases MMP-1,-2,-3,-9,-13) markers of inflammation were examined. RESULTS: Multinucleated giant cells were present in all specimens containing suture material but not in the control specimens. No significant differences were found in the number of giant cells between the intact suture groups at either time point. Significantly higher numbers of giant cells were noted in the particles C group compared to the intact suture C group at both time points (p = 0.021 at 1 week, p = 0.003 at 4 weeks). Quantitative analysis of immunohistochemical staining expression at 4 weeks showed that significantly more MMP (-1,-2,-9,-13) was expressed in the particles C group than the intact suture C group (p = 0.024, p = 0.009, p = 0.002, and p = 0.007 for MMP-1, MMP-2, MMP-9, and MMP-13, respectively). No significant difference was seen in the expression of MMP-3 (p = 0.058). CONCLUSIONS: There were no differences observed between the biological reactivity of commonly used intact orthopaedic sutures A, B, and C. However, wear particles of suture C elicited a significantly greater inflammatory response than intact suture alone. This was confirmed by increased numbers of multinucleated giant cells as well as MMP ( -1,-2,-9,-13) expression. Further studies are needed to determine whether this inflammatory response may play a role in the development of post-arthroscopic glenohumeral chondrolysis or interfere with biological healing. These findings have important clinical implications relating to surgical technique and surgical implant design.

Application of Calcium Sulfate for Dead Space Management in Soft Tissue: Characterisation of a Novel In Vivo Response.

Management of dead space (DS) is a fundamental aspect of surgery. Residual DS following surgery can fill with hematoma and provide an environment for bacterial growth, increasing the incidence of postoperative infection. Materials for managing DS include polymethyl-methacrylate (PMMA), which is nonresorbing and requires removal in a second surgical procedure. The use of calcium sulfate (CS) offers the advantage of being fully absorbed and does not require subsequent surgical removal. As CS has historically been used as a bone void filler, there are some concerns for the risk of heterotopic ossification (HO) when implanted adjacent to soft tissue. This study assessed the osteoinductive potential of CS and identified and characterised residual material present in muscle tissue using histology, energy-dispersive X-ray spectroscopy analysis, and scanning electron microscopy (SEM). CS beads with and without antibiotic were implanted in intramuscular sites in both athymic rats and New Zealand white rabbits. At 28 days after implantation in the rat model, no signs of osteoinduction were observed. In the rabbit model, at 21 days after implantation, almost complete bead absorption and presence of a "halo" of material in the surrounding muscle tissue were confirmed. Our results suggested that the halo of material was a calcium phosphate precipitate, not HO.

Critical Size Bone Defect Healing Using Collagen-Calcium Phosphate Bone Graft Materials.

The need for bone graft materials to fill bony voids or gaps that are not related to the intrinsic stability of the bone that arise due to trauma, tumors or osteolysis remains a clinically relevant and significant issue. The in vivo response of collagen-tricalcium phosphate bone graft substitutes was evaluated in a critical size cancellous defect model in skeletally mature rabbits. While the materials were chemically virtually identical, new bone formation, implant resorption and local in vivo responses were significantly different. Differences in the in vivo response may be due, in part, collagen source and processing which influences resorption profiles. Continued improvements in processing and manufacturing techniques of collagen-tricalcium phosphate bone graft substitutes can result in osteoconductive materials that support healing of critical size bone defects even in challenging pre-clinical models.

Evaluation of Transbronchial Lung Cryobiopsy Size and Freezing Time: A Prognostic Animal Study.

BACKGROUND: Transbronchial lung biopsy using a cryoprobe is a novel way of sampling lung parenchyma. Correlation of freezing time with biopsy size and complications has not been evaluated in vivo. OBJECTIVES: The primary aim of the study is to evaluate the correlation between transbronchial cryobiopsy freezing time and size. The secondary aims are to evaluate histological quality of the biopsy and evaluate procedure-associated complications. METHODS: Transbronchial lung cryobiopsies were obtained from two anaesthetised sheep using a 1.9-mm cryoprobe inserted into a flexible bronchoscope under fluoroscopic guidance. Freezing times ranged from 1 to 6 s (n = 49). The cryobiopsies were evaluated histologically with respect to their size and quality. Complications of bleeding and pneumothorax were recorded. RESULTS: The mean cross-sectional area of the cryobiopsy ranged from 4.7 ± 2.1 to 15.7 ± 15.3 mm2. There was a significant positive correlation between increasing freezing time and cryobiopsy cross-sectional area (p = 0.028). All biopsies contained lung tissue with preserved parenchyma. Crush and freeze artefacts were not observed and tissue architecture was intact in all specimens. Small blood vessels and terminal bronchioles were observed in 88% of specimens. All cryobiopsies caused nil or mild haemorrhage with the exception of only 1 episode of severe haemorrhage at 6 s freezing time. Pneumothoraces occurred at 2, 5 and 6 s freezing time and required chest tube insertion. The most significant haemorrhage and pneumothoraces occurred at 5 and 6 s. Our results suggest an initial freezing time of 3 s can provide the maximal biopsy size while minimising major complications. CONCLUSION: The optimal transbronchial cryobiopsy freezing time is initially 3 s. This time is associated with minimal complications and large artefact-free biopsies.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Development of a Novel Model for the Assessment of Dead-Space Management in Soft Tissue.

Following extensive surgical debridement in the treatment of infection, a "dead space" can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1ß indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.