Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration.

Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy.

Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture).

The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration.

Acknowledgments

This research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd.

Abstract

Introduction

Closed avulsion of the Flexor Digitorum Profundus (FDP) from distal phalanx most commonly affects the ring finger when an extensive force is applied to a finger in active flexion. Whilst it is undoubtably reasonable to provide treatment for those who present with symptoms, there may be a cohort of people who sustain an avulsion without noticing. This study aims to quantify the effect of ring finger FDP avulsion on overall grip strength to determine the functional effect of a missed injury

Bone grafts are frequently used to augment bone healing. Autologous bone graft is the gold standard for osteogenesis but is limited by availability and donor site morbidity. The processing required to lower the immunogenicity of allograft also reduces the osteogeneic properties. Bone marrow contains mesenchymal stem cells (MSCs) which differentiate into osteoblasts, forming bone. Our study examined the use of bone marrow to enhance the osteogenic properties of allograft.

Bioactive proteins within allogenic bone graft stimulate marrow-derived MSCs to differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft.

After informed consent, bone marrow aspirates were taken from five patients during orthopaedic operations. Freeze-dried ethylene oxide treated allograft, from a number of donors, was obtained from the bone bank. MSCs isolated from each marrow aspirate were grown on eight samples of test allograft. Further allograft was heated to 70°C to denature the osteogenic proteins and MSCs from each aspirate were grown on 8 samples, as a negative control. Osteoblastic differentiation of MSCs cultured on the types of allograft was compared.

Scanning electron microscopy confirmed that MSCs covered the allograft after 14 days. Transmission electron microscopy showed that cells on the test allograft were characteristic of osteoblasts and produced collagen extracellular matrix. The levels of osteoblastic proteins, ALP, osteopontin and Type I pro-collagen, produced by cells on test allograft were significantly greater compared with heat-treated control (P< 0.005), after days 7 and 14.

Our study showed that marrow-isolated MSCs could be successfully cultured on allograft. As the levels of osteoblastic proteins increased significantly when MSCs were grown on allograft, osteogenic proteins within allograft caused MSCs to change into osteoblasts. This confirms that autologous marrow MSCs could be grown on allograft to increase its osteogenic prior to grafting, resulting in increased rate of bony healing.

To compare the actual with the reported incidence of pressure sores to determine the accuracy of data (classification errors) and completeness of data (differences between manual and computer generated figures), retrospective data was collected regarding pressure sore rates following primary elective total hip arthroplasty operations carried out in 2001. Pressure sores rates were noted by nursing staff and entered into a computer database.

Four consultant orthopaedic surgeons were involved, across 2 sites – 1 NHS (PRH) and 1 local private hospital.

Preliminary audit reports indicated an alarmingly high pressure sore rate across the two units – 17/172 (9.9%) PRH and 23/71 (32.4%) private hospital.

Two major errors were revealed. In terms of accuracy of data, grade 1 areas (erythema without active ulceration) were included at both sites. These are only potential sites of pressure sores and should not have been used to calculate actual pressure sore rate. In terms of completeness of data, manual verification of the number of operations performed revealed a discrepancy between the theatres’ logbook entries and private unit computer figures. 97 rather than 71 operations were performed. There was no such discrepancy at the NHS site.

The data was reanalysed to obtain the actual pressure sore rate. For the NHS unit, grade 1areas were subtracted, causing the rate to fall from 32.4% to 1.0%. The two errors caused a dramatic and significant difference between reported and actual pressure sore rate.

Poor data collection leads to inaccurate audit, leading to inappropriate management. The concern is that similar errors, accumulated across key complication targets and specialities, will have a profound impact on NHS star ratings.

It is likely that league tables will to some extent determine hospital finance in the future. The major indicator used in league table calculations in orthopaedics is mortality rates following surgery. Therefore, our study audited the accuracy of mortality data.

A previous audit of our department by an external audit company was found to show an apparent excess mortality rate, due to the company’s failure to distinguish between true operations and certain procedures, i.e. urethral catheterisation. We were concerned that these flawed results may find their way into the publicised tables of the Department of Health (DH). We thus audited deaths in 2000/1 and compared the results with DH data.

DH league table figures combine the mortality numbers for all surgical specialities. Our analysis was based on DH criteria [www.doh.gov.uk/performancer-atings/2001), death within 30 days of operation, following non-elective admission and excluded certain procedures. PAS was used to identify deaths and all case notes were reviewed.

From review of the notes, the criteria for post-operative death were fulfilled by 54/131 deaths (41%). By speciality, these included 14/33 deaths in orthopaedics, general surgery (25/73) and neurosurgery (15/25). The DH identified 64 post-operative deaths in this period. DH calculations were applied to compare our postoperative mortality results (54 deaths) with those of the DH (64 deaths). Although there was no significant difference between our observed death rate and the DH’s, using our results the hospital’s ranking improved from twelfth to sixth place in 42 small acute hospitals.

The observed mortality rate in our hospital is very close to that published by the DH and the national average. From the results of our study, we are confident that the flawed data from the external company did not enter the system and distort the DH’s league tables.

Therefore, hospitals should not wast money on audits by external companies.

Introduction: Wear particle induced osteolysis is one of the main reasons for revision total hip replacements (THRs). Loss in bone stock as a result of aseptic loosening is responsible for inferior results in revision THRs. Results from impaction grafting to fill osteolytic defects are frequently inconsistent. Our hypothesis is that the combination of autologous mesenchymal stem cells (MSCs) and allograft will enhance bone regeneration. This study asks whether: MSCs with allograft scaffolds survive at a normal impaction force during revision THRs.

Method: MSCs were isolated from a sheep iliac crest aspirate, expanded in culture and seeded onto irradiated sheep allografts (n=9). Viability of MSCs was assayed with alamar blue with absorbance measured on day 4 (before impaction). The constructs were then impacted using forces 3, 6, and 9 kN extrapolated in surgery then assayed daily for 6 days. The control was 0 kN. Samples were resin embedded after 10 days for histology and pieces of graft were taken for scanning electron microscopy (SEM).

Results: The 0KN control shows an MSC growth curve with a lag period and log phase. Compared with the control, the 3 and 6 kN showed initial reduction in cell proliferation measured by alamar blue (^p=0.015, ^p=0.002) but recovered by day 8, while 9kN showed a significant reduction (^p=0.011) over the time (Figure 1).

For cell proliferation over time, 3 and 6 kN showed no differences, but 9 kN showed a significant difference between day 4 and day 8 (^p=0.031). SEM and histological analysis showed a network of cuboidal cells on the allograft surface.

Conclusions: The results showed that MSCs recovered from impaction of 3 and 6 kN after an initial reduction in metabolism and exceeded original cell seeding densities with no significant difference in proliferation. Viability of MSCs were not effected by impaction forces up to 6 kN. This study shows that stem cells mixed with allograft are a potential method for repairing bone defects in revision total hip replacements.

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.

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 1cm³ 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.

Introduction: Bone grafts are frequently used in orthopaedic operations to augment bone healing. Autologous bone graft is the gold standard for osteogenesis, but the amount available from the patient’s iliac crest is often insufficient to fill the defect and donor site morbidity is a significant complication. Alternatively, allograft can be implanted into patients, however, processing is necessary to reduce the immunicity of the graft and the risk of transmission of infection, but this destroys osteoprogenitor cells and hence reduces the osteogenic properties of the graft. Mesenchymal stem cells (MSCs) are present in bone marrow and have the ability to differentiate into osteoblasts. Therefore our study examined the use of MCSs, from bone marrow, to enhance the osteogenic properties of allograft.

Hypothesis: MSCs cultured on freeze-dried ethylene oxide treated bone allograft differentiate into osteoblasts, thereby increasing the osteogenic nature of the graft material.

Method: After informed consent, bone marrow aspirates were taken from 10 patients during elective orthopaedic operations. MSCs were characterized using Stro-1 antibody and grown on freeze-dried ethylene oxide treated bone allograft in vitro.

The hypothesis was tested on three groups of graft, with eight samples in each group. Firstly, freeze-dried ethylene oxide treated bone graft was tested (group 2). For a negative control, allograft was heated to 70°C to denature the osteogenic proteins (group 1). The final group tested the effect of additional osteogenic supplements (100nM dexamethasone, 0.05mM ascorbic acid and 10mM (-glycerol phosphate) on MSCs on allograft (group 3).

Osteoblastic differentiation of MSCs was observed under scanning (SEM) and transmission (TEM) electron microscopy, and by measuring protein levels: alkaline phosphatase (ALP), osteopontin and type I pro-collagen over 14 days.

Results: SEM confirmed that MSCs could be successfully cultured on bone allograft. Cells grown in groups 2 and 3 were characteristic of metabolically active osteoblasts and collagen extracellular matrix was observed under TEM. The amount of ALP protein produced by MSCs cultured in groups 2 and 3 increased significantly over 14 days (P< 0.05), but there was no increase in group 1. ALP, osteopontin and type I pro-collagen production was significantly greater for group 2 than for group 1 and for group 3 than for group 2 (P< 0.05).

Discussion and Conclusions: ALP, type I pro-collagen and osteopontin proteins are known to be produced by osteoblasts during increasing cell maturation and the levels of each of these proteins increased significantly when MSCs were cultured on allograft for 14 days compared with the negative control. The addition of osteogenic supplements significantly increased production of these proteins. Furthermore, MSCs cultured in groups 2 and 3 produced extracellular collagen matrix. These results are consistent with allograft causing MSCs to differentiate into osteoblasts and that this differentiation increases with additional osteogenic supplements.

This study confirms that MSCs, derived from autologous bone marrow, could be used to increase the osteogenic potential of allograft, thereby increasing bony healing in patients.

Mesenchymal stem cells (MSCs) are pluripotential cells present in marrow, which have the ability to differentiate into osteoblasts, chrondrocytes and adipocytes. Potential skeletal tissue engineering uses include healing bone defects, spinal fusion and revision arthoplasty surgery. A means of storing viable mesenchymal stem cells is necessary in order for these cells to be readily available for clinical use. The aim of this study was to determine whether cryopreservation has any effect on the osteogenic potential of human bone marrow derived MSCs.

Five normal iliac crest bone marrow aspirates were obtained following informed consent from patients. Each aspirate was divided into two equal samples. Ficoll-separation was used to isolate the MSCs. The fresh MSCs from one sample were cryopreserved, while the other was cultured as a control population. To assess the osteogenic potential of the MSCs after cryopreservation a sample of cells from each population was cultured with osteogenic supplements and the increase in alkaline phosphatase (ALP) and osteocalcin production was compared.

Cryopreservation was not observed to effect the primary cultures of MSCs, which became confluent after a similar period in culture (12–14 days), forming colonies with recognized MSCs morphology. The expression of ALP and osteocalcin after stimulating the MSCs to differentiate with osteogenic supplements, was not significantly altered by the cryopreservation process (P> 0.05).

In conclusion MSCs obtained from fresh human bone marrow aspirates can be cryopreserved without compromise to their proliferation rate or osteogenic potential, confirming that this is a useful means of storing viable cells for future clinical use.

Osteoblast progenitor cells can be isolated from human bone marrow and on an appropriate carrier following differentiation into osteoblasts a bone block could be formed. This supply of autologous, osteoinductive bone graft substitute would have significant implications for clinical use. The aim of the study was to assess whether osteoblast progenitor cells isolated from human bone marrow, seeded onto porous hydroxyapatite (HA) blocks adhere, proliferate and differentiate into osteoblasts under the influence of HA alone.

After informed consent, bone marrow was aspirated from the iliac crest of 8 patients. The osteoblast progenitor cells were separated from the haematological cells and cultured in vitro. Evidence for the osteoblast progenitor nature of the cells was obtained by adding osteogenic supplements: dexamethasone, ascorbic acid and b-glycophosphate, and comparing alkaline phosphatase (ALP) and osteocalcin expression with that of unstimulated cells. Undifferentiated osteoblast progenitor cells were seeded at a density of 2x10 ⁶ cells/porous HA cylindrical block (8 x 8 x10 mm). The cell adhesion to the HA was observed, and proliferation and ALP expression was measured over 15 days.

In monolayer culture the isolated bone marrow cells were morphologically identified as mesenchymal stem cells. When osteogenic supplements were added the phenotype became consistent with the morphology of osteoblastic cells, and the ALP expression was significantly higher (P< 0.05) after 5 days in culture compared with cells that had not been stimulated to differentiate.

On the HA osteoblast progenitor cells were adherent and became more osteoblastic, being separated from the HA surface by an osteoid matrix layer on electron microscopy. The ALP expression by these cells increased significantly (P< 0.05) over the 15 day culture period.

Bone marrow contains mesenchymal stem cells with osteogenic potential that are known as osteoblast progenitor cells. In this study we have shown that osteoblast progenitor cells can be isolated from human bone marrow and will adhere to and proliferate on HA blocks in vitro, and differentiate into osteoblasts spontaneously under the influence of the HA scaffold. These constructs could be used as osteoinductive bone grafts.