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Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 47 - 47
17 Nov 2023
Algarni M Amin A Hall A
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Abstract

Objectives

Osteoarthritis (OA) is a painful and debilitating disorder of diarthroidal joints. Progressive degeneration of the cartilage extracellular matrix (ECM) together with abnormal chondrocyte characteristics occur leading to a switch to a fibroblast-like phenotype and production of mechanically-weak cartilage. Early changes to chondrocytes within human cartilage have been observed including chondrocyte swelling[1] together with the development of thin cytoplasmic processes which increase in number and length with degeneration[2]. Changes to chondrocyte phenotype in degenerate cartilage are associated with F-actin redistribution and stress fibres (SF) formation, leading to morphologically-dedifferentiated (fibroblast-like) chondrocytes[3,4]. It is unclear if these processes are a consequence of ‘passive’ cell swelling into a defective ECM or an ‘active’ event driven by changes in cell metabolism resulting in alterations to cell shape. To address this, we have quantified and compared the distribution and levels of F-actin, a key cytoskeletal protein involved in the formation of cytoplasmic processes, within in situ chondrocytes in non-degenerate and mildly degenerate human cartilage.

Methods

Human femoral head cartilage was obtained from 21 patients [15 females, 6 males, average age 69.6yrs, (range 47–90yrs)] following femoral neck fracture, with Ethical Approval and patient's permission. Cartilage explants were removed from areas graded non-degenerate grade 0 (G0) or mildly degenerate grade 1 (G1) and cultured for up to 3wks in Dulbecco's Modified Eagle's Medium (DMEM) +/− 25% human serum (HS). In situ chondrocytes were stained with CMFDA (5-chloromethylfluoresceindiacetate, Cell-Tracker Green®) and phalloidin (F-actin labelling) and imaged by confocal microscopy and analysed quantitatively using ImageJ and Imaris® software.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 24 - 24
17 Nov 2023
Alturkistani Z Amin A Hall A
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Abstract

Objective

The preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective.

Methods

Ex vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_16 | Pages 56 - 56
17 Nov 2023
Algarni M Amin A Hall A
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Abstract

Objectives

Osteoarthritis (OA) is a complex joint disorder characterised by the loss of extracellular matrix (ECM) leading to cartilage degeneration. Changes to cartilage cell (chondrocyte) behaviour occur including cell swelling, the development of fine cytoplasmic processes and cell clustering leading to changes in cell phenotype and development of focal areas of mechanically-weak fibrocartilaginous matrix[1]. To study the sequence of events in more detail, we have investigated the changes to in situ chondrocytes within human cartilage which has been lightly scraped and then cultured with serum.

Methods

Human femoral heads were obtained with Ethical permission and consent from four female patients (mean age 74 yrs) undergoing hip arthroplasty following femoral neck fracture. Osteochondral explants of macroscopically-normal cartilage were cultured as a non-scraped control, or scraped gently six times with a scalpel blade and both maintained in culture for up to 2wks in Dulbecco's Modified Eagle's Medium (DMEM) with 25% human serum (HS). Explants were then labelled with CMFDA (5-chloromethylfluorescein-diacetate) and PI (propidium iodide) (10μM each) to identify the morphology of living or dead chondrocytes respectively. Explants were imaged using confocal microscopy and in situ chondrocyte morphology, volume and clustering assessed quantitatively within standardised regions of interest (ROI) using Imaris® imaging software.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 62 - 62
17 Apr 2023
Herren A Luczak A Amin A Hall A
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Early changes within articular cartilage during human idiopathic osteoarthritis are poorly understood. However alterations to chondrocyte morphology occur with the development of fine cytoplasmic processes and cell clusters, potentially playing a role in cartilage degeneration. The aggrecanase ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs-4) has been implicated as an important factor in cartilage degradation, so we investigated the relationship between chondrocyte morphology and levels of ADAMTS-4 in both non-degenerate and mildly osteoarthritic human cartilage.

Human femoral heads were obtained following consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants of normal (grade 0; G0) and mildly osteoarthritic (grade 1; G1) cartilage were labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate). Explants were cryosectioned (30μm sections), and labelled for ADAMTS-4 by fluorescence immunohistochemistry. Sections were imaged with confocal microscopy, allowing the semi-quantitative analysis of ADAMTS-4 and 3D visualisation of in situ cell morphology.

With cartilage degeneration from G0 to G1, there was a decrease in the proportion of chondrocytes with normal rounded morphology (P<0.001) but an increase in the proportion of cells with processes (P<0.01) and those in clusters (P<0.001;[4(1653)]; femoral heads:cells). Although average levels of ADAMTS-4 for all cells was the same between G0 and G1 (P>0.05), a change was evident in the distribution curves for cell-specific ADAMTS-4 labelling. Cell-by-cell analysis showed that ADAMTS-4 levels were higher in chondrocytes with cytoplasmic processes compared to normal cells (P=0.044) however cells in clusters had lower levels than normal cells (P=0.003;[3(436)]). Preliminary data suggested that ADAMTS-4 levels increased with larger chondrocyte clusters.

These results suggest complex heterogeneous changes to levels of cell-associated ADAMTS-4 with early cartilage degeneration – increasing in cells with processes and initially decreasing in clusters. Increased levels of ADAMTS-4 are likely to produce focal areas of matrix weakness potentially leading to early cartilage degeneration.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 39 - 39
17 Apr 2023
Saiz A O'Donnell E Kellam P Cleary C Moore X Schultz B Mayer R Amin A Gary J Eastman J Routt M
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Determine the infection risk of nonoperative versus operative repair of extraperitoneal bladder ruptures in patients with pelvic ring injuries. Pelvic ring injuries with extraperitoneal bladder ruptures were identified from a prospective trauma registry at two level 1 trauma centers from 2014 to 2020. Patients, injuries, treatments, and complications were reviewed. Using Fisher's exact test with significance at P value < 0.05, associations between injury treatment and outcomes were determined.

Of the 1127 patients with pelvic ring injuries, 68 (6%) had a concomitant extraperitoneal bladder rupture.

All patients received IV antibiotics for an average of 2.5 days. A suprapubic catheter was placed in 4 patients. Bladder repairs were performed in 55 (81%) patients, 28 of those simultaneous with ORIF anterior pelvic ring. The other 27 bladder repair patients underwent initial ex-lap with bladder repair and on average had pelvic fixation 2.2 days later. Nonoperative management of bladder rupture with prolonged Foley catheterization was used in 13 patients. Improved fracture reduction was noted in the ORIF cohort compared to the closed reduction external fixation cohort (P = 0.04).

There were 5 (7%) deep infections. Deep infection was associated with nonoperative management of bladder rupture (P = 0.003) and use of a suprapubic catheter (P = 0.02). Not repairing the bladder increased odds of infection 17-fold compared to repair (OR 16.9, 95% CI 1.75 – 164, P = 0.01).

Operative repair of extraperitoneal bladder ruptures substantially decreases risk of infection in patients with pelvic ring injuries. ORIF of anterior pelvic ring does not increase risk of infection and results in better reductions compared to closed reduction. Suprapubic catheters should be avoided if possible due to increased infection risk later. Treatment algorithms for pelvic ring injuries with extraperitoneal bladder ruptures should recommend early bladder repair and emphasize anterior pelvic ORIF.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_9 | Pages 78 - 78
17 Apr 2023
Luczak A Battle I Amin A Hall A
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The development of cytoplasmic processes from in situ chondrocytes is a characteristic feature of early osteoarthritis in human cartilage. The processes involve cytoskeletal elements and are distinct from the short primary cilia described in human chondrocytes. Vimentin is an intermediate filament playing an essential structural and signal-transduction role. We determined cellular levels and distribution of vimentin in chondrocytes of different morphologies in non-degenerate and mildly osteoarthritic cartilage.

Femoral heads were obtained after consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants were graded as non-degenerate (grade 0;G0) or mildly osteoarthritic (grade 1;G1) and labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate) for cell shape. Explants were cryosectioned and labelled for vimentin by fluorescence immunohistochemistry. In situ chondrocyte morphology was identified by confocal microscopy as either normal (rounded/elliptical) or abnormal (with one or more cytoplasmic process of ≥2µm) and vimentin levels and distribution determined semi-quantitatively and related to chondrocyte morphology.

When all cells in G0 and G1 cartilage were compared, there was no difference between average levels of vimentin per cell (P=0.144)[6(261)];femoral heads:cells). When cells were separated on the basis of morphology, there was no difference between vimentin levels in cells with one or more cytoplasmic process compared to those of normal morphology (P>0.05;[6(261)]). However vimentin levels were much greater at the base of cytoplasmic processes compared to distant areas of the same cells (P=0.021)[5(29)]).

Although overall levels of chondrocyte vimentin do not change in these early stages of osteoarthritis, the formation and structure of these substantial chondrocyte cytoplasmic processes involves changes to its distribution. These morphological changes are similar to those occurring during chondrocyte de-differentiation to fibroblasts reported in osteoarthritis which results in the formation of mechanically-inferior fibro-cartilage. Alterations to chondrocyte vimentin distribution either directly or indirectly may play a role in cartilage degeneration.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 142 - 142
11 Apr 2023
Algarni M Amin A Hall A
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Cartilage degeneration and loss are key events in the initiation and progression of osteoarthritis (OA). Changes to chondrocyte volume and morphology (in the form of cytoplasmic processes) and thus cell phenotype are implicated, as they lead to the production of a mechanically-weakened extracellular matrix. The chondrocyte cytoskeleton is intimately linked to cell volume and morphology and hence we have investigated alterations to levels and distribution of chondrocyte F-actin that occur during early OA.

The femoral heads (FH) from hip joints (N=16) were obtained with ethical permission and patient consent following femoral neck fracture. Cartilage was assessed as grade 0 (non-degenerate) and grade 1 (superficial fibrillation) using OARSI criteria. In situ chondrocyte volume and F-actin distribution were assessed using the fluorescent indicators (5-chloromethyl fluorescein diacetate (CMFDA)) and phalloidin, and imaged and quantified by confocal microscopy, ImarisTM and ImageJ software.

There were no differences between the volume or total F-actin levels of in situ chondrocytes within the superficial zone of grade 0 (n=164 cells) compared to grade 1 (n=145) cartilage (P>0.05). However, a more detailed analysis of phalloidin labelling was performed, which demonstrated significant increases in both intense punctuate (IP) or intense areas (IA) (P<0.0001; P=0.0175 respectively). A preliminary analysis of IP and IA F-actin labelling suggested that while the former did not appear to be associated with changes to chondrocyte morphology, most of the cytoplasmic processes were associated with the presence of IA at the starting point of the protrusion.

These results demonstrate marked changes to F-actin distribution in chondrocytes in the very early stages of cartilage degeneration as occurs in OA. These subtle changes are probably an early indication of a change to the chondrocyte phenotype and thus worthy of further study as they may lead to deleterious alterations to matrix metabolism and ultimately cartilage weakening.


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_8 | Pages 118 - 118
11 Apr 2023
Styczynska-Soczka K Cawley W Samuel K Campbell J Amin A Hall A
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Articular cartilage has poor repair potential and the tissue formed is mechanically incompetent. Mesenchymal stromal cells (MSCs) show chondrogenic properties and the ability to re-grow cartilage, however a viable human model for testing cartilage regeneration and repair is lacking. Here, we describe an ex vivo pre-clinical femoral head model for studying human cartilage repair using MSCs.

Human femoral heads (FHs) were obtained following femoral neck fracture with ethical permission/patient consent and full-depth cartilage wells made using a 3mm biopsy punch. Pancreas-derived mesenchymal stromal cells (P-MSC) were prepared in culture media at ~5000 cells/20µl and added to each well and leakage prevented with fibrin sealant. After 24hrs, the sealant was removed and medium replaced with StemProTM chondrogenesis differentiation medium. The FHs were incubated (37oC;5% CO2) for 3wks, followed by a further 3wks in standard medium with 10% human serum with regular medium changes throughout. Compared to wells with medium only, A-MSCs produced a thin film across the wells which was excised en-block, fixed with 4% paraformaldehyde and frozen for cryo-sectioning.

The cell/tissue films varied in thickness ranging over 20-440µm (82±21µm; mean±SEM; N=3 FHs). The thickness of MSC films abutting the cartilage wells was variable but generally greater (15-1880µm) than across the wells, suggesting an attachment to native articular cartilage. Staining of the films using safranin O (for glycosaminoglycans; quantified using ImageJ) was variable (3±8%; mean±SEM; N=3) but in one experiment reached 20% of the adjacent cartilage. A preliminary assessment of the repair tissue gave an O'Driscoll score of 10/24 (24 is best).

These preliminary results suggest the ex vivo femoral head model has promise for studying the capacity of MSCs to repair cartilage directly in human tissue, although optimising MSCs to produce hyaline-like tissue is essential.

Supported by the CSO (TCS/17/32).


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_2 | Pages 10 - 10
1 Mar 2021
Kooiman E Styczynska-Soczka K Amin A Hall A
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Abstract

Objectives

Human articular cartilage chondrocytes undergo changes to their morphology and clustering with cartilage degeneration as occurs in osteoarthritis(1). The consequences of chondrocyte de-differentiation on mechanically-resilient extracellular matrix metabolism are, however, unclear. We have assessed whether there is a relationship between abnormal chondrocyte morphology, as demonstrated by the presence of cytoplasmic processes, and chondrocyte clustering, with cell-associated type-I collagen during cartilage degeneration.

Methods

The femoral heads of 9 patients were obtained (with Ethical permission/consent) following hip replacement surgery and cartilage areas graded (Grade-0 non-degenerate; Grade-1 mildly degenerate). In situ chondrocyte morphology and cell-associated type-I collagen were labelled fluorescently with CMFDA Cell tracker green, and immuno-fluorescence respectively then visualised/quantified using confocal laser scanning microscopy and imaging software.


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_2 | Pages 6 - 6
1 Mar 2021
Styczynska-Soczka K Amin A Simpson H Hall A
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Abstract

Objectives

The development of promising therapeutics for cartilage repair/regeneration have been hampered by the inadequacy of existing animal models and lack of suitable translational ex-vivo human tissue models. There is an urgent unmet need for these to assess repair/regenerative (orthobiologic) treatments directly in human tissue. We describe methodology allowing the successful long-term ex-vivo culture of non-degenerate whole human femoral heads that may be used as a model for testing new orthobiologic therapies.

Methods

Fifteen fresh, viable human femoral heads were obtained from 15 patients (with ethical permission/consent) undergoing hemiarthroplasty for hip fracture, and cultured aseptically (37°C) for up to 10wks. Culture conditions included static/stirred standard media (Dulbecco's modified Eagle's medium; DMEM) and supplementation with 10% human serum (HS). Chondrocyte viability, density, cell morphology, cell volume, glycosaminoglycan(GAG)/collagen content, surface roughness and cartilage thickness were quantified over time.


Orthopaedic Proceedings
Vol. 95-B, Issue SUPP_13 | Pages 19 - 19
1 Mar 2013
Prys-Jones O Amin A Hall A
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The internal fixation of osteochondral fragments in fractures normally utilizes intra-articular screws inserted through a pilot hole drilled into cartilage/bone. This trauma causes cartilage injury leading to chondrocyte death. We have quantified the cell death following cartilage drilling and identified irrigation conditions that can protect chondrocytes. Articular cartilage of bovine metacarpophalangeal joints of 3yr-old cows was irrigated in the presence/absence of saline of various compositions. Holes were then made using a standard 1.5mm drill (Ortho Solutions Ltd.) at 18,000 rpm through the articular cartilage into bone. Osteochondral explants were then harvested and cultured in Dulbecco's Modified Eagle's Medium containing chloromethylfluorescein-di-acetate and propidium iodide (10uM each), to label living chondrocytes green and dead cells red, respectively. Axial images were taken by confocal microscopy and the width of the zone of cell death (ZCD) around the hole determined. With no irrigation, new drills caused a ZCD of 171±25um, which was increased when drills used 50+ times were tested (279±31um;p=0.03). With saline irrigation, the ZCD was reduced for old drills (150±6um;p=0.016) but not for new drills (124±8um) suggesting the heating effect of the old drills caused additional chondrocyte death. However for new drills, the ZCD was further reduced significantly to 82±7um when the osmolarity of the saline irrigation solution was raised to 480mOsm using sucrose. Data are mean±s.e.m., from at least 5 separate experiments each with a minimum of 3 replicates. The results demonstrate a chondroprotective effect of raising the osmolarity of saline used during drilling of cartilage which could be clinically beneficial.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 24 - 24
1 Aug 2012
McLintock B Banfield C Amin A Hall A
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Saline (0.9%) is typically used to rinse joints during osteo-articular surgery. It is not unusual for cartilage to then be exposed to the air of the operating theatre for 1-2hrs, which can lead to chondrocyte death. We have compared the survival of in situ chondrocytes within bovine cartilage which has been rinsed in various solutions or simply drained of synovial fluid (SF) and then allowed to dry, to identify approaches that could reduce chondrocyte death arising from cartilage drying.

Metacarpophalangeal joints from 3yr-old cows were opened under aseptic conditions. The joints were then (a) rinsed with saline (Baxter's Healthcare, Newbury), (b) rinsed with saline+glucose (20mM; both 300mOsm) or (c) drained of SF, and allowed to dry at room temperature. Full depth cartilage explants were taken after 2hrs, placed into Dulbecco's modified Eagle's medium and incubated with CMFDA (5-chloromethyl-fluorescein diacetate; 10microM) and propidium iodide (10microM) for the identification/quantification of living and dead cells respectively by confocal scanning laser microscopy and image analysis.

After 2hrs, the appearance and properties of the cartilage of the drying joints were clearly different. Saline-rinsed cartilage was dark purple and appeared dull with the cartilage difficult to sample. However when the rinsing solution was saline+glucose, or when joints were drained of SF, the cartilage was almost identical to the freshly-opened joint with a pearly-blue, shiny appearance, and cartilage sampling was easy.

Chondrocyte death was markedly increased in saline rinsed/dried joints after 2hrs (21±9% cell death). In contrast, there was no significant (P>0.05) death in saline+glucose rinsed/dried (2±1%) or SF-drained joints (3±2%;means±s.e.m.;n=5). The loss of cartilage wet weight over 2hrs (time=0 taken as 100%) was almost identical between cartilage rinsed in saline (73.6±1.6%), saline + glucose (78.6±1.1%) or SF (75.0±0.2%; data means±s.d.;n=2).

These results suggest that it was not the loss of water per se during cartilage drying that was the key determinant of chondrocyte viability. As chondrocytes are normally anaerobic, the rise in cartilage pO2 which occurs during exposure to air could have a deleterious effect on cell viability however the presence of glucose or SF protects through an anti-oxidant effect.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 79 - 79
1 Aug 2012
Houston D Amin A White T Hall A
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Intra-articular screw fixation is indicated for internal fixation of large osteochondral fragments secondary to trauma or osteochondritis dissecans. During surgery, orthopaedic drills are used to prepare a hole through which the screw can pass. Previous work has shown that mechanical injury to articular cartilage results in a zone of cell death adjacent to the traumatised articular cartilage (1). Here, we characterise and quantify the margin of in situ chondrocyte death surrounding drill holes and screws (standard cortical and headless compression designs) placed in mature bovine articular cartilage to model the orthopaedic procedure.

Drill holes (1mm) were made through the articular cartilage and bone of intact bovine metacarpophalangeal joints obtained from 3-yr old cows within 12hrs of slaughter. Osteochondral explants (∼1cm square and 2-3mm thick) encompassing the drilled holes in articular cartilage and subchondral bone were harvested using a chisel. Explants were then incubated in Dulbecco's modified Eagle's medium for 45mins with CMFDA (5-chloromethylfluorescein diacetate) and PI (propidium iodide; both at 10micromolar) to identify/quantify living and dead in situ chondrocytes respectively in a consecutive series of axial optical sections using confocal scanning laser microscopy (CLSM).

The drill holes through cartilage appeared to have clearly defined edges with no macroscopic evidence of cartilage splitting. However visualisation of fluorescently-labelled in situ chondrocytes by CLSM demonstrated clear cell death around the periphery of the drilled hole which was 166±19 micrometers in width. This increased with a larger diameter (1.5mm) drill to 450±151 micrometers (all data are means±s.e.m.; n=3). Preliminary experiments indicated that the margin of chondrocyte death around a 1.5mm hole was dramatically increased further by the insertion of screws into pre-drilled holes.

These results suggest that the mechanical trauma associated with cartilage drilling and the insertion of intra-articular screws occurs with marked death of in situ chondrocytes extending into normal cartilage beyond the area occupied by the screw. As chondrocytes are not replaced in mature cartilage, their loss around the hole/screw will mean that the extracellular matrix is not maintained, inevitably leading to cartilage failure.