Yield optimisation and molecular characterisation of uncultured CD271+ mesenchymal stem cells in the Reamer Irrigator Aspirator waste bag.

Bone reconstruction requires the use of autografts from patients' iliac crest (IC); for large-volume defects bone void fillers and autologous mesenchymal stem cells (MSCs) are often added. The Reamer/Irrigator/Aspirator (RIA) device provides the means of harvesting large amounts of autograft and additionally yields a waste bag containing MSCs, which is currently discarded. The aim of this study was to enumerate and characterise native MSCs from RIA waste bag and compare them to 'gold-standard' donor-matched MSCs from IC bone marrow (BM). IC-BM from age matched trauma patients was used as control. In RIA waste bags the median MSC yield established using a colony-forming fibroblast assay was 314333 (range 5 x 104-1.4 x 106), equivalent to approximately one litre of IC-BM aspirate. CD271+ cells were present at high levels in RIA waste bags, had MSC surface phenotype (CD90+CD73+CD105+CD34>sup>-CD61-CD19-CD31-CD33-) and expressed genes associated with multipotentiality, osteogenesis, adipogenesis and angiogenic support. RIA- CD271+ MSCs were transcriptionally similar to donor-matched IC-CD271+ MSCs (76 % transcripts); with the majority of bone-related and Wnt pathway molecules being expressed at comparable levels. Lower-level expression of MCAM/CD146 and 5/13 hypoxia-related molecules was found in RIA-CD271+ MSCs, potentially reflecting their native residence in a more hypoxic environment of the endosteum and bone cortex. These data suggest that long bones contain very large numbers of MSCs, transcriptionally-similar to IC-BM MSCs; they can be procured by reaming using the RIA device and used, following concentration, as autologous and potentially allogeneic bone repair therapy.

Complex Tibial Shaft Fractures in Children Involving the Distal Physis Managed with the Ilizarov Method.

INTRODUCTION: Segmental fractures in the juvenile distal tibia with physeal involvement present specific challenges. Injury to the growth plate may be overlooked, potentially resulting in late sequelae. Fracture stabilization can be complex. Previous reports of management of such an injury are by open reduction and internal fixation. This study reviews the management and outcome of a group of such patients treated with Ilizarov external fixators. MATERIALS AND METHODS: Patients aged 16 or younger treated in our unit between March 2013 and November 2014 by Ilizarov circular fine wire fixation for tibial fractures with ipsilateral physeal injuries were identified. Retrospective collection of patient demographics, fracture classification, treatment pathways, fixation methods, postoperative follow-up, outcomes, and complications was undertaken. RESULTS: Eight patients were identified; two had Gustilo and Anderson grade IIIA open injuries. All were managed definitively using an Ilizarov external fixator in combination with percutaneous screw fixation of the physeal component as required. All patients were ambulant during treatment and were allowed unrestricted weight-bearing immediately postoperative. All but one attended school. All fractures united. In follow-up, one patient had a distal tibial physeal growth arrest, but there were no other complications. CONCLUSION: Pediatric patients with complex distal tibial fractures should be scrutinized for concomitant physeal injury. Where identified treatment, using a combination of internal fixation and an Ilizarov fixator can be considered. HOW TO CITE THIS ARTICLE: Rogers GP, Tan HB, Foster P, et al. Complex Tibial Shaft Fractures in Children Involving the Distal Physis Managed with the Ilizarov Method. Strategies Trauma Limb Reconstr 2019;14(1):20-24.

MRI appearances of the femur following bone graft harvesting using the Reamer-Irrigator-Aspirator.

The reamer-irrigator-aspirator is increasingly being used to harvest autologous bone graft from the femur. The purpose of this study was to investigate the extent of neo-vascularisation and new bone formation that occurs within the medulla following the procedure, and determine if new bone formation would potentially allow a repeat bone harvest in those individuals subsequently requiring further bone graft. Eleven patients who had undergone femoral bone harvest were examined with MRI. The nature of the tissue within the medulla and the extent of neo-vascularisation were assessed. MRI was performed between 3 months and 28 months following bone graft harvest, mean 14 months. Intense vascularisation of the endostial cortical surface and neo-vascularisation of the haematoma within the canal occurred as soon as 3 months following bone harvest. From as early as 14 months the tissue was replaced by normal intramedullary bone. The formation of new bone within the medullary canal gives the potential for a repeat reaming, should further bone graft be required at a later date.

Examining the feasibility of clinical grade CD271+ enrichment of mesenchymal stromal cells for bone regeneration.

INTRODUCTION: Current clinical trials utilize mesenchymal stromal cells (MSCs) expanded in culture, however these interventions carry considerable costs and concerns pertaining to culture-induced losses of potency. This study assessed the feasibility of new clinical-grade technology to obtain uncultured MSC isolates from three human intra-osseous tissue sources based on immunomagnetic selection for CD271-positive cells. MATERIALS AND METHODS: MSCs were isolated from bone marrow (BM) aspirates or surgical waste materials; enzymatically digested femoral heads (FHs) and reamer irrigator aspirator (RIA) waste fluids. Flow cytometry for the CD45-/lowCD73+CD271+ phenotype was used to evaluate uncultured MSCs before and after selection, and to measure MSC enrichment in parallel to colony forming-unit fibroblast assay. Trilineage differentiation assays and quantitative polymerase chain-reaction for key transcripts involved in bone regeneration was used to assess the functional utility of isolated cells for bone repair. RESULTS: Uncultured CD45-/lowCD271+ MSCs uniformly expressed CD73, CD90 and CD105 but showed variable expression of MSCA-1 and SUSD2 (BM>RIA>FH). MSCs were enriched over 150-fold from BM aspirates and RIA fluids, whereas the highest MSC purities were obtained from FH digests. Enriched fractions expressed increased levels of BMP-2, COL1A2, VEGFC, SPARC and CXCL12 transcripts (BM>RIA>FH), with the highest up-regulation detected for CXCL12 in BM (>1300-fold). Following culture expansion, CD271-selected MSCS were tri-potential and phenotypically identical to plastic adherence-selected MSCs. DISCUSSION: A CD271-based GMP-compliant immunomagnetic selection resulted in a substantial increase in MSC purity and elevated expression of transcripts involved in bone formation, vascularisation and chemo-attraction. Although this technology, particularly from RIA fluids, can be immediately applied by orthopaedic surgeons as autologous therapy, further improvements in MSC purities and pre-clinical testing of product safety would be required to develop this process for allogeneic applications.

Induced periosteum a complex cellular scaffold for the treatment of large bone defects.

OBJECTIVE: Surgically induced periosteal membrane holds great potential for the treatment of large bone defects representing a simple alternative to combinations of exogenous stem cells, scaffolds and growth factors. The purpose of this study was to explore the biological basis for this novel regenerative medicine strategy in man. METHODS: Eight patients with critical size defects were treated with the induced membrane (IM) technique. After membrane formation 1cm(2) biopsy was taken together with matched, healthy diaphyseal periosteum (P) for comparative analysis. Morphological characteristics, cell composition and growth factor expression were compared. Functional and molecular evaluation of mesenchymal stromal cell (MSC) activity was performed. RESULTS: Both tissues shared similar morphology although IM was significantly thicker than P (p=0.032). The frequency of lymphocytes, pericytes (CD45(-)CD34(-)CD146(+)) and cells expressing markers consistent with bone marrow MSCs (CD45(-/low)CD271(bright)) were 31. 3 and 15.5-fold higher respectively in IM (all p=0.043). IM contained 3-fold more cells per gramme of tissue with a similar proportion of endothelial cells (CD45(-)CD31(+)). Expressed bone morphogenic protein 2, vascular endothelial growth factor and stromal derived factor 1 (SDF-1) are key tissue regeneration mediators. Adherent expanded cells from both tissues had molecular profiles similar to bone marrow MSCs but cells from IM expressed greater than 2 fold relative abundance of SDF-1transcript compared to P (p=0.043). CONCLUSION: The IM is a thick, vascularised structure that resembles periosteum with a cellular composition and molecular profile facilitating large defect repair and therefore may be described as an "induced-periosteum". This tissue offers a powerful example of in situ tissue engineering.