Background

Replacing bone lost as a consequence of trauma or disease is a major challenge in the treatment of musculoskeletal disorders. Tissue engineering strategies seek to harness the potential of stem cells to regenerate lost or damaged tissue. Bone marrow aspirate (BMA) provides a promising autologous source of skeletal stem cells (SSCs) however, previous studies have demonstrated that the concentration of SSCs required for robust tissue regeneration is below levels present in iliac crest BMA, emphasising the need for cell enrichment strategies prior to clinical application.

Recent approaches have sought to harness the potential of stem cells to regenerate bone lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of skeletal stem cells (SSCs) for such applications, however previous studies have demonstrated that the concentration of SSCs present in iliac crest BMA is below that required for robust bone regeneration. Here we present a novel acoustic-facilitated filtration strategy to concentrate BMA for SSCs, clinically applicable for intra-operative orthopaedic use.

The aim of this study was to demonstrate the efficacy of this strategy in concentrating SSCs from iliac crest bone marrow, as well as femoral canal BMA from older patients.

Iliac crest BMA (Lonza, Rockville, MD, USA) and femoral canal BMA was obtained with informed consent from older patients during total hip replacement. 5 to 40ml of BMA was processed via the acoustically-aided exclusion filtration process to obtain 2-8 fold volume reductions. SSC concentration and function was assessed by flow-cytometry, assays for fibroblastic colony-forming units (CFU-F) and multi-lineage differentiation along chondrogenic, osteogenic and adipogenic pathways examined. Seeding efficiency of enriched and unprocessed BMA (normalised to cell number) onto allograft was assessed.

Iliac crest BMA from 15 patients was enriched for SSCs in a processing time of only 15 minutes. Femoral BMA from 15 patients in the elderly cohort was concentrated up to 5-fold with a corresponding enrichment of viable and functional SSCs, confirmed by flow cytometry and assays for CFU-F. Enhanced osteogenic (P<0.05) and chondrogenic (P<0.001) differentiation was observed using concentrated aspirate, as evidenced by biochemical assay and semi-quantitative histological analysis. Furthermore, enhanced cell seeding efficiency onto allograft was seen as an effect of SSC concentration per ml of aspirate (P<0.001), confirming the utility of this approach for application to bone regeneration.

The ability to rapidly enrich BMA demonstrates potential for intra-operative application to enhance bone healing and offers immediate capacity for clinical application to treat many scenarios associated with local bone stock loss. Further in vivo analysis is ongoing prior to clinical tests.

Introduction: Articular cartilage has limited capacity for regeneration. Tissue engineering strategies offer future hope for cartilage replacement and repair. In an attempt to mimic functional native cartilage for tissue repair, current research focuses on construct/implant designs that simulate an embryonic like microenvironment to promote cellular differentiation along a chondrogenic lineage. The aim of the present study was, for the first time, to illustrate the differences between human neonatal and adult chondrocytes along with bone marrow stromal cells (HBMSCs) to differentiate the factors that promote chondrogenesis and maintain functional homeostasis.

Material and Methods: Adult chondrocytes, neonatal chondrocytes and HBMSCs were cultured in monolayers for 1, 2 and 3 weeks in basal or chondrogenic media. Expression of transcription factor Sox9, Aggrecan (ACAN) and Collagen type II (COL2A)was compared via real time polymerase chain reaction (q-PCR). Alternatively, cells were seeded onto 3D PLGA scaffolds and cultured in vitro for 3 and 6 weeks in basal or chondrogenic media. Paraffin sections of the constructs were stained with Alcian blue/ Sirius red and expression of Collagen type II and Aggrecan was visualised via immunohistochemistry.

Results: For monolayer cultures of all three cell types, at week 1, expression of all three genes was down regulated in basal medium compared to levels in chondrogenic medium. By week 2, q-PCR revealed an increased expression of Col2A in chondroinduced neonatal chondrocytes compared to adult chondrocytes and HBMSCs. A steady increase in SOX9 expression was observed with time in all three cell types in chondrogenic medium. However, SOX9 expression in week 2 was higher for each cell type in basal medium compared with chondrogenic medium. ACAN expression by HBMSCs was greatly enhanced compared with that of neonatal and adult chondrocytes after 2 weeks in chondrogenic medium. By week 3, basal cultures of all cell types showed an overall lower level of gene expression compared with chondroinduced cells. 3D constructs revealed the formation of cartilage like tissue for all three cell types with the presence of a prominent superficial layer and middle zone in the chondroinduced constructs. A superficial layer was also observed in constructs cultured in basal media but there was no evidence of any other characteristic zones. A fibrous capsule had formed around the chondroinduced tissue by week 6. Thinnest capsules were observed for constructs seeded with neonatal cells, with thickest capsules in constructs seeded with HBMSCs. Immunohistochemistry revealed a greater presence of aggrecan and type II collagen in the chondroinduced constructs compared to those cultures in basal media.

Conclusion: This comparative study indicates a major difference between the microenvironment of human neonatal chondrocytes, adult chondrocytes and HBMSCs. The expression of high amounts of COL2A and ACAN (considered to be middle to late markers in chondrogenesis) in week 1 in neonatal chondrocytes indicates a difference in temporal gene expression during chondrogenesis or in maintaining cartilage homeostasis. The study provides potentially useful information to inform cell-based therapies for cartilage regeneration.

Articular cartilage has limited regenerative potential. Regeneration via autografts or cell therapy is clinically efficacious but the extent of regenerative success depends upon use of an appropriate cell source. The aim of this study was to compare the proliferative and chondrogenic potentials of three human cell types (human bone marrow stromal cells - HBMSCs, neonatal and adult chondrocytes) commonly used in cartilage tissue engineering.

HBMSCs, neonatal and adult chondrocytes (passage 2) were cultured in basal and chondrogenic media. At 2, 4 and 6 days, the cells were analysed for morphology and doubling time. Alkaline phosphatase specific activity (ALPSA) was quantified for each group at 2, 4 and 6 weeks. Chondrogenic potential of each cell type was assessed via a pellet culture model. Cryosections were stained with Alcian blue/Sirius Red.

HBMSCs showed either elongated or polymorphic phenotypes, with a doubling time of 40 h. Neonatal chondrocytes showed a uniform spindle shape and had the shortest doubling time (16 h). Adult chondrocytes, were also spindle shaped, though slightly larger than the neonatal cells, with a longer doubling time of 22 h. Expression of ALPSA in basal media was of the order HBMSCs > adult chondrocytes > , neonatal chondrocytes. In chondrogenic culture, this order changed to adult chondrocytes > HBMSCs > neonatal chondrocytes. In 3D pellet cultures, all three cell types stained positive for Alcian Blue and showed the presence of chondrocyte-like cells enclosed in lacunae.

This comparative study suggests that neonatal chondrocytes are the most proliferative with lowest ALP expression. However, in terms of clinical applications, HBMSCs may be better for cartilage regeneration given their lower ALP expression under chondrogenic conditions when compared with adult chondrocytes under the same conditions. The study has provided information to inform clinical cell therapy for cartilage regeneration.

Aim: To evaluate the necessity for further radiological investigation in patients with suspicion of rotatory subluxation of the atlanto-axial complex on plain radiography following acute cervical trauma. To outline guidelines for assessment of patients with atlanto-axial asymmetry on plain radiography.

Methods: A retrospective review of all patients who had undergone atlanto-axial CT scanning as a result of radiographic C1–C2 asymmetry following cervical spine trauma in the 3 year period from January 1999 to December 2001. The plain X-ray and CT images were reviewed retrospectively and correlated with their clinical presentation and outcome by the senior author.

Results: Twenty-eight patients were included in the study. Acute cervical spine trauma had occurred most commonly following a road traffic accident. No patient was found to have acute cervical spine torticollis or severe cervical pain. Patients age ranged from 21–44 years (M:F – 15:13). All patients were found to have atlanto-odontoid asymmetry on initial plain X-ray. No patients were found to have rotatory subluxation on CT images. 3 patients were found to have minor degrees (< 10°) of rotation on the CT scan which is within normal limits. 9 patients (32%) were found to have congenital odontoid lateral mass asymmetry. All patients were treated conservatively and had no further intervention. All plain radiographs were then assessed to determine the underlying reason for asymmetry. In 19 cases the orientation of the radiographic beam in combination with head rotation was found to be at fault.

Conclusion: Rotatory subluxation of the cervical spine is a rare but serious condition in the adult. The condition is suspected radiologically in the presence of odontoid lateral mass asymmetry on open mouth view. The application of ATLS principles in the initial assessment of trauma patients has resulted in a significant increase in the number of radiological examinations performed. This has led inevitably to an increase in the number of anomalies identified. An average of 400 c-spine X-rays per year are performed for trauma in our casualty department. In this study, we have identified 9 patients out of a total of 29 with congenital odontoid lateral mass asymmetry over a 3 year period. This represents approximately 0.75% of the cervical spine X-rays and should be considered in the differential diagnosis following acute cervical trauma. We outline guidelines for recognising benign atlanto-axial asymmetry.