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Bone & Joint Research
Vol. 3, Issue 5 | Pages 139 - 145
1 May 2014
Islam K Dobbe A Komeili A Duke K El-Rich M Dhillon S Adeeb S Jomha NM

Objective

The main object of this study was to use a geometric morphometric approach to quantify the left-right symmetry of talus bones.

Methods

Analysis was carried out using CT scan images of 11 pairs of intact tali. Two important geometric parameters, volume and surface area, were quantified for left and right talus bones. The geometric shape variations between the right and left talus bones were also measured using deviation analysis. Furthermore, location of asymmetry in the geometric shapes were identified.

Results

Numerical results showed that talus bones are bilaterally symmetrical in nature, and the difference between the surface area of the left and right talus bones was less than 7.5%. Similarly, the difference in the volume of both bones was less than 7.5%. Results of the three-dimensional (3D) deviation analyses demonstrated the mean deviation between left and right talus bones were in the range of -0.74 mm to 0.62 mm. It was observed that in eight of 11 subjects, the deviation in symmetry occurred in regions that are clinically less important during talus surgery.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 65 - 65
1 Sep 2012
Adesida A Sernik J Croutze R Laouar L Secretan C Jomha NM
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Purpose

Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold.

Method

Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 66 - 66
1 Sep 2012
Adesida A Matthies N Sierra A Jomha NM
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Purpose

The biomechanical role of the meniscus in the knee joint is a function of its extracellular matrix which consists of type I collagen throughout, type II collagen in the inner meniscus region and glycosaminoglynated (GAG) proteins of which aggrecan is the most prevaleet. Meniscus reparative capacity is limited, particularly when a defect is located in the inner avascular portion, and menisectomy predisposes the joint to osteoarthritis. Using meniscus cells in tissue engineering strategies has been advocated to generate functional meniscus substitutes. However, meniscus cells, like chondrocytes of cartilage, lose their matrix-forming phenotype during culture expansion. Co-culture of chondrocytes with stem cells has been shown to result in enhanced matrix formation. We hypothesized that meniscus cells in co-culture with stem cells will result in increased matrix formation.

Method

Tissue specimens were obtained after approval of the local ethical committee and informed consent. Menisci were obtained from 3 patients undergoing total knee arthroplasty; (53–84; mean age 66.6). Meniscus cells were isolated after digestion of menisci with collagenase II. Isolated meniscus cells were plated for 24–48 hr before use. Bone marrow aspirates were obtained from the iliac crest of 3 donors: 1 female (46) and 2 males (15 and 21) undergoing routine orthopaedic procedures. Plastic adherent bone marrow stromal cell populations were isolated and expanded under normal oxygen tension of 21%O2 in a-MEM growth media plus FGF-2 until passage 2. Cells were mixed at a variety of meniscus cells (Men): BMSC ratio including 5/95, 10/90 and 25/75, respectively. Mixed cells were centrifuged to form spherical pellets followed by culture in a defined serum free chondrogenic differentiation medium. Control groups were pure Men and pure BMSCs. Total cell number per pellet was 25×104. Pellets were cultured for 3 weeks under normal oxygen tension. Thereafter, pellets were processed: biochemically for GAG and DNA content, and histologically for Safranin-O staining of sulphated GAG and immunohistochemical analyses for collagen types I and II. Analysis was performed on a minimum of 2 independent pellets.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 62 - 62
1 Sep 2012
Adesida A Sierra A Jomha NM
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Purpose

Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2.

Method

Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 563 - 563
1 Nov 2011
Barley RD Bagnall KM Jomha NM
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Purpose: Traditionally, chondrocyte growth and characterization studies have been conducted using non-physiologic, normoxic, monolayer culture systems that have the major drawback of dedifferentiation. Recently, however, the use of novel 3D culture systems, cytokine supplementation or hypoxic culturing techniques have shown that chondrocyte dedifferentiation can be greatly reduced. Unfortunately, to date, no single culture technique has been identified that completely prevents the dedifferentiation-related changes in ECM gene expression. We hypothesized that combining a high density culture condition with an hypoxic environment would improve chondrocyte phenotype retention as determined by gene expression and protein production when compared to current standard culture conditions.

Method: Freshly isolated normal human articular chondrocytes were maintained in three culture conditions:

conventional monolayer culture,

high density monolayer culture (HDMC) and

embedded in alginate and compared to freshly isolated positive controls (FIC) and fibroblasts as negative controls.

The conventional monolayer cultures were harvested at confluence while HDMC and alginate-embedded chondrocytes (AEC) were maintained in culture for 8 weeks. Parallel experiments were conducted under normoxic (21% O2) and hypoxic (5% O2) conditions for all three experimental groups. Chondrocytes were harvested, RNA was extracted and quantitative RT-PCR was performed using primers for collagens (I, II, VI, IX and XI), aggrecan, SOX-9, HIF-1, 3 different integrins and GAPDH. In addition, collagen and GAG content was quantified when possible using Sircol and Blyscan assays respectively.

Results: HDMC cultures in hypoxic conditions showed a 2.5 fold increase in wet weight, a 6.9 fold increase in GAG content and a 1.3 fold increase in collagen content relative to normoxic HDMCs. With respect to gene expression levels, only the HDMCs in hypoxic culture conditions yielded mRNA expression levels of collagen II, IX, XI, aggrecan, HIF-1, SOX-9 and one Integrin that were consistent with the levels seen in freshly isolated chondrocytes (positive control). Importantly, HDMC culture in hypoxic conditions also yielded the lowest levels of collagen I of any experimental condition.

Conclusion: This research demonstrated that high density monolayer culture in hypoxic conditions prevented the severe loss of chondrocyte phenotype typically associated with conventional monolayer culture. Cells cultured in these conditions demonstrated gene expression levels similar to those seen in FICs, which are superior to those seen following conventional culture conditions such as the use of alginate beads. These culture conditions provide a novel opportunity to maintain chondrocyte phenotype over a prolonged period of time while generating extracellular matrix that may be beneficial for treatment of full thickness cartilage defects.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 244 - 245
1 Jul 2011
Manolescu AR Cinats D Secretan C O’Neill D Cheeseman C Bagnall K Jomha NM
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Purpose: Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological mechanism and full characterization of each individual stage through which the BM-MSC differentiate into adult chondrocytes is not yet understood. The physiological micro-environment of the chondrocytes is intensely hypoxic which triggers over-expression SLC2A proteins (GLUTs) in their membranes as a compensatory mechanism for energy production within the glycolytic cycle.

Method: We cultured and differentiated BM-MSC, and adult chondrocytes in hypoxic (5% O2 tension) and normoxic (20% O2) conditions. Within this cell populations we screened for the presence of the 12 GLUT genes as well as quantification of the variation of the 12 GLUTs gene translation by simple pcr and rt-pcr. The expression profile of the GLUT proteins was investigated using western blot analysis and immunohistochemistry. Functional characterization of the GLUTs expressed in the different cell populations was carried out by the means of radio-isotope labeled hexose fluxes done accordingly to the substrate specificity and kinetic properties particular to each SLC2A isoforms.

Results: Our data showed that the functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSC comprised an extensive expression of fructose-transporting GLUTs as opposed to the glucose-only transporting isoforms expression in normoxic BM-MSC. The flux data showed clear similarities in functional GLUT profiles between BM-MSC cultured in hypoxic conditions, adult chondrocytes. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-gluose, 3-orthomethyl-glucose and galactose) in these cellular populations under both hypoxic and normoxic conditions and in the presence and absence of Cytochalasin B (a GLUT1-specific inhibitor) showed that SLC2A class II transporters (GLUTs 5, 7, 9 and 11) play a more important role in the uptake of sugars by the normal hypoxic chondrocytes when compared to the ubiquitously-expressed GLUT1.

Conclusion: Use of this approach allows the correct culturing conditions to be identified that would select for those chondrocyte precursors from the total BM-MSC population that would have the best potential for producing viable articular cartilage. In addition, specific substrates for GLUTs isoforms could be used for physiologic, non-invasive and real time imaging of cartilage, BM-MSC and cartilage autograft by means of Positron Emission Tomography.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 245 - 245
1 Jul 2011
Maragh K Bater J Secretan C Bagnall KM Jomha NM
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Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time.

Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with real time PCR, probing specifically for the genes responsible for the production of collagen I, collagen II, aggrecan, and GAPDH. This was done in duplicate.

Results: Collagen II gene expression was maintained over seven weeks in cells left at confluence but was decreased in passaged cells. Collagen I gene expression decreased over seven weeks in cells left at confluence, but remained the same in passaged cells. Aggrecan gene expression remained the same in both groups.

Conclusion: Current culture and expansion techniques that employ passaging (as used in clinical scenarios) result in significant alterations in gene expression that are inconsistent with the current definition of a “chondrocyte”. Culturing chondrocytes at confluence can produce gene expression more similar to native chondrocytes but even these cells have expression of collagen type I that should not be present in chondrocytes. The results of this study suggest that further investigation is required to develop chondrocyte culture and expansion techniques that minimize the de-differentiation of chondrocytes by maintaining collagen II gene expression and eliminating/preventing collagen I gene expression.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_III | Pages 243 - 244
1 Jul 2011
Secretan C Bater J Bagnall K Jomha NM
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Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration.

Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes. Real time polymerase chain reaction was utilized to quantify and compare the essential markers, collagen II, collagen I and aggrecan, in the stem cell derived chondrocytes. The CD44 enriched and CD44 depleted populations were compared.

Results: The cells isolated possessed a cell morphology and surface antigen profile consistent with a MSC population. In addition, both experimental groups demonstrated multipotent ability. Real time PCR analysis of the chondrogenic cells demonstrated that the CD44 positive population expressed collagen II and aggrecan at a significantly higher level than the CD44 negative population.

Conclusion: To date no group has successfully identified a relationship between a MSC subpopulation and the multipotent progenitors responsible for generating cartilage. This work demonstrated that there are MSC sub-populations with different potential for chondrogenic expression and represents an important step towards identifying MSC subpopulations with enhanced cartilage formation potential.


The Journal of Bone & Joint Surgery British Volume
Vol. 81-B, Issue 5 | Pages 775 - 779
1 Sep 1999
Jomha NM Pinczewski LA Clingeleffer A Otto DD

Deficiency of the anterior cruciate ligament (ACL) is a common disorder which can lead to changes in lifestyle. We followed 59 patients who had had arthroscopic reconstruction of the ACL using a central-third patellar-tendon autograft for seven years to assess the long-term effectiveness of recent advances in reconstruction of the ACL. The standard criteria for evaluation of the International Knee Documentation Committee, the Lysholm knee score and measurements using the KT 1000 arthrometer all showed satisfactory results. Deterioration in the clinical performance after seven years was associated with osteoarthritic changes and correlated with chronic ligament injuries and meniscectomy. There were three traumatic and three spontaneous ruptures.

We believe that the procedure can be successful, but remain concerned about failure of the graft and osteoarthritis. The results raise questions about the best time to operate and suggest that early surgery may reduce the risk of osteoarthritis.


The Journal of Bone & Joint Surgery British Volume
Vol. 80-B, Issue 5 | Pages 781 - 784
1 Sep 1998
Borton DC Lucas P Jomha NM Cross MJ Slater K

Rupture of the tendons of both peroneus longus and peroneus brevis results in considerable disability. We have performed transfer of flexor digitorum longus (FDL) to peroneus brevis in two patients with lateral instability of the hindfoot due to chronic transverse tears of both tendons for which end-to-end repair was not possible. Both patients had excellent function when reviewed after eight and six years, respectively, with no symptoms. CT showed a normal appearance of the FDL in both patients, but the peroneal muscles looked abnormal. Transfer of the FDL provides a reliable solution to lateral instability of the hindfoot resulting from loss of function of both peronei.