Helen S. McCarthy

RANK, RANKL and osteoprotegerin in bone biology and disease

Upon the discovery of RANK, RANKL and OPG in the late 1990s, their importance in the maintenance of the skeletal structure and their dramatic role in bone disease were largely unexpected. In recent years the understanding of these proteins, in particular their regulation, has greatly increased. This review aims to bring the interested reader up to date with the latest news and views on the mechanisms controlling bone resorption in normal and pathological conditions.

Increased circulating Dickkopf-1 in Paget's disease of bone

UNLABELLED Dickkopf-1 (Dkk-1) is a secreted inhibitor of Wnt signaling which in adults regulates bone turnover. Dkk-1 over-production is implicated in osteolytic disease where it inhibits bone formation and stimulates bone breakdown. Recently it was reported that osteoblastic cells from Pagets disease of bone (PDB) over-expressed Dkk-1. OBJECTIVE To see if increased Dkk-1 was detected in serum from patients with PDB. RESULTS Dkk-1 and total serum alkaline phosphatase activity (tsAP) were significantly elevated in sera from PDB patients. Patients with polyostotic PDB had significantly higher levels of tsAP but not Dkk-1, than monostotic patients. TsAP but not Dkk-1, was significantly lower in sera from bisphosphonate treated versus untreated PDB patients. Dkk-1 and tsAP were not significantly correlated. CONCLUSIONS Dkk-1 may be a useful biomarker of PDB and we speculate that Dkk-1 may play a central role in the etiology of PDB.

Fourier Transform Infrared Imaging and Infrared Fiber Optic Probe Spectroscopy Identify Collagen Type in Connective Tissues

Hyaline cartilage and mechanically inferior fibrocartilage consisting of mixed collagen types are frequently found together in repairing articular cartilage. The present study seeks to develop methodology to identify collagen type and other tissue components using Fourier transform infrared (FTIR) spectral evaluation of matrix composition in combination with multivariate analyses. FTIR spectra of the primary molecular components of repair cartilage, types I and II collagen, and aggrecan, were used to develop multivariate spectral models for discrimination of the matrix components of the tissues of interest. Infrared imaging data were collected from bovine bone, tendon, normal cartilage, meniscus and human repair cartilage tissues, and composition predicted using partial least squares analyses. Histology and immunohistochemistry results were used as standards for validation. Infrared fiber optic probe spectral data were also obtained from meniscus (a tissue with mixed collagen types) to evaluate the potential of this method for identification of collagen type in a minimally-invasive clinical application. Concentration profiles of the tissue components obtained from multivariate analysis were in excellent agreement with histology and immunohistochemistry results. Bone and tendon showed a uniform distribution of predominantly type I collagen through the tissue. Normal cartilage showed a distribution of type II collagen and proteoglycan similar to the known composition, while in repair cartilage, the spectral distribution of both types I and II collagen were similar to that observed via immunohistochemistry. Using the probe, the outer and inner regions of the meniscus were shown to be primarily composed of type I and II collagen, respectively, in accordance with immunohistochemistry data. In summary, multivariate analysis of infrared spectra can indeed be used to differentiate collagen type I and type II, even in the presence of proteoglycan, in connective tissues, using both imaging and fiber optic methodology. This has great potential for clinical in situ applications for monitoring tissue repair.

A histological comparison of the repair tissue formed when using either Chondrogide® or periosteum during autologous chondrocyte implantation

OBJECTIVE In this study, we compare the clinical and histological outcome between periosteum and Chondrogide(®) during autologous chondrocyte implantation (ACI). METHOD This study consisted of 88 patients having received ACI in the knee; 33 treated with Chondrogide(®) (ACI-C) and 55 with periosteum (ACI-P). Post-operative biopsies were taken at a mean of 16.6 ± 8 months (range 7-37 months) and 19 ± 18.4 months (range 4-114) for ACI-C and ACI-P respectively. Histological assessment was performed using the ICRS II and OsScore scoring systems. The immunolocalisation of elastin and collagen types I and II was analysed using specific antibodies. Lysholm scores, a measure of knee function, were obtained pre- and post-operatively at the time of biopsy and annually thereafter. RESULTS Compared with ACI-P, the repair tissue formed from patients treated with ACI-C demonstrated a significantly higher score for cellular morphology (ICRS II score), significantly better surface morphology from medial femoral condyle treated defects (ICRS II score) and a significantly higher proportion of hyaline cartilage formation (OsScore). Elastin fibres were present in both ACI-C and ACI-P samples, although their presence was very variable in quantity, distribution, orientation, thickness and length. Patients treated with ACI-C demonstrated significantly more collagen type II immunolocalisation compared with ACI-P. Both groups exhibited a significant increase in Lysholm score post-ACI. CONCLUSIONS This study demonstrates a significantly better quality of repair tissue formed with ACI-C compared with ACI-P. Hence Chondrogide(®) is perhaps a better alternative to periosteum during ACI.

Platelet-derived growth factor stimulates osteoprotegerin production in osteoblastic cells.

Osteoprotegerin (OPG) is a major regulator of osteoclastogenesis, bone resorption and vascular calcification. OPG is produced by various cell types including mesenchymally derived cells, in particular, osteoblastic cells. Here we show OPG production by osteoblastic cells was stimulated by platelet‐derived growth factor (PDGF) in two human osteosarcoma cell lines (MG63, Saos‐2), a mouse pre‐osteoblastic cell line (MC3T3‐E1) and human bone marrow stromal cells (hMSC) by 152%, 197%, 113% and 45% respectively over 24 h. OPG was measured in the cell culture medium by immunoassay. PDGF isoforms AA, BB and AB show similar stimulation of OPG production. Message for OPG was also increased similarly to the increased secretion into the culture medium. Using specific inhibitors of cell signalling we demonstrate that PDGF acts through the PDGF receptor, PKC, PI3K, ERK and P38 and not via NF‐kB or JNK. The importance of PDGF in fracture healing suggests a role for OPG production in countering bone resorption during the early phase of this process. J. Cell. Physiol. 218: 350–354, 2009.

Dickkopf-1 as a potential therapeutic target in Paget's disease of bone

Importance of the field: Wnt signalling plays a role in maintaining healthy bone mass. Dickkopf-1 (DKK-1) is a soluble inhibitor of Wnt signalling and its excessive expression contributes to bone loss in rheumatoid arthritis and multiple myeloma. New therapeutics have been developed for treatment of these conditions that target DKK-1 expression. DKK-1 is elevated in serum of patients with Pagets disease of the bone (PDB) and evidence is accumulating for a role of DKK-1 in PDB. Areas covered in this review: The role of Wnt signalling and DKK-1 in bone health and disease and the aetiology of PDB in the light of recent advances in understanding of Wnt signalling. What the reader will gain: PDB is a disorder of unknown aetiology characterised by localised increase in unregulated bone remodelling resulting in osteolytic and osteosclerotic lesions. Evidence is adduced for the involvement of Wnt signalling, DKK-1 and osteoblasts in PDB pathogenesis. Take home message: At present there is no cure for PDB and the current treatment of choice are bisphosphonates. These treat the resorptive phase of PDB but do not prevent its return. We present a new perspective on the aetiology of PDB and speculate on DKK-1 as a therapeutic target.

Chondrogenic Potency Analyses of Donor-Matched Chondrocytes and Mesenchymal Stem Cells Derived from Bone Marrow, Infrapatellar Fat Pad, and Subcutaneous Fat

Autologous chondrocyte implantation (ACI) is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs). In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM), infrapatellar fat pad (FP), and subcutaneous fat (SCF), compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies.

Evaluating Joint Morbidity after Chondral Harvest for Autologous Chondrocyte Implantation (ACI): A Study of ACI-Treated Ankles and Hips with a Knee Chondral Harvest.

Objective To establish if harvesting cartilage to source chondrocytes for autologous chondrocyte implantation (ACI) results in donor site morbidity. Design Twenty-three patients underwent ACI for chondral defects of either the ankle or the hip. This involved cartilage harvest from the knee (stage I), chondrocyte expansion in the laboratory and implantation surgery (stage II) into the affected joint. Prior to chondral harvest, no patient had sought treatment for their knee. Lysholm knee scores were completed prior to chondral harvest and annually post-ACI. Histological analyses of the donor site were performed at 12.3 ± 1.5 months for 3 additional patients who had previously had ACI of the knee. Results The median preoperative Lysholm score was 100, with no significant differences observed at either 13.7±1.7 months or 4.8±1.8 years postharvest (median Lysholm scores 91.7 and 87.5, respectively). Patients whose cartilage was harvested from the central or medial trochlea had a significantly higher median Lysholm score at latest follow-up (97.9 and 93.4, respectively), compared with those taken from the intercondylar notch (median Lysholm score 66.7). The mean International Cartilage Repair Society (ICRS) II histological score for the biopsies taken from the donor site of 3 additional knee ACI patients was 117 ± 10 (maximum score 140). Conclusions This study suggests that the chondral harvest site in ACI is not associated with significant joint morbidity, at least up to 5 years postharvest. However, one should carefully consider the location for chondral harvest as this has been shown to affect knee function in the longer term.

Mesenchymal stromal cells derived from whole human umbilical cord exhibit similar properties to those derived from Wharton's jelly and bone marrow

Mesenchymal stromal cells (MSC) can be isolated from several regions of human umbilical cords, including Whartons jelly (WJ), artery, vein or cord lining. These MSC appear to be immune privileged and are promising candidates for cell therapy. However, isolating MSC from WJ, artery, vein or cord lining requires time‐consuming tissue dissection. MSC can be obtained easily via briefly digesting complete segments of the umbilical cord, likely containing heterogenous or mixed populations of MSC (MC‐MSC). MC‐MSC are generally less well characterized than WJ‐MSC, but nevertheless represent a potentially valuable population of MSC. This study aimed to further characterize MC‐MSC in comparison to WJ‐MSC and also the better‐characterized bone marrow‐derived MSC (BM‐MSC). MC‐MSC proliferated faster, with significantly faster doubling times reaching passage one 8.8 days sooner and surviving longer in culture than WJ‐MSC. All MSC retained the safety aspect of reducing telomere length with increasing passage number. MSC were also assessed for their ability to suppress T‐cell proliferation and for the production of key markers of pluripotency, embryonic stem cells, tolerogenicity (CD40, CD80, CD86 and HLA‐DR) and immunomodulation (indoleamine 2,3‐dioxygenase [IDO] and HLA‐G). The MC‐MSC population displayed all of the positive attributes of WJ‐MSC and BM‐MSC, but they were more efficient to obtain and underwent more population doublings than from WJ, suggesting that MC‐MSC are promising candidates for allogeneic cell therapy in regenerative medicine.

Autologous Bone Plug Supplemented With Autologous Chondrocyte Implantation in Osteochondral Defects of the Knee

Background: Structural and functional outcome of bone graft with first- or second-generation autologous chondrocyte implantation (ACI) in treating cartilage and subchondral bone defect has not been reported previously. Purpose: To evaluate the outcome of simultaneous transplantation of an autologous bone plug with first- or second-generation ACI for restoration of concomitant subchondral bone and full-thickness cartilage defect in the femoral condyle of the knee. Study Design: Case series; Level of evidence, 4. Methods: Seventeen patients (mean ± SD age, 27 ± 7 years; range, 17-40 years)—12 with osteochondritis dissecans (International Cartilage Repair Society [ICRS] grades 3 and 4) and 5 with an isolated osteochondral defect (ICRS grade 4)—had the defect reconstructed with implantation of a unicortical autologous bone graft combined with ACI (the OsPlug technique). Functional outcome was assessed with Lysholm scores obtained preoperatively and at 1 and 5 years postoperatively. The repair site was evaluated with the Oswestry Arthroscopy Score (OAS), MOCART score (magnetic resonance observation of cartilage repair tissue), and ICRS II histology score. Formation of a subchondral lamina and lateral integration of the bone grafts were evaluated from magnetic resonance imaging scans. Results: The mean defect size was 4.5 ± 2.6 cm2 (range, 1-9 cm2), and the mean depth was 11.3 ± 5 mm (range, 5-18 mm). The preoperative Lysholm score improved from 45 (interquartile range [IQR], 24; range, 16-79) to 77 (IQR, 28; range, 41-100) at 1 year (P = .001) and 70 (IQR, 35; range, 33-91) at 5 years (P = .009). The mean OAS of the repair site was 6.2 (range, 0-9) at a mean of 1.3 years. The mean MOCART score was 61 ± 22 (range, 20-85) at 2.6 ± 1.8 years. Histology demonstrated generally good integration of the repair cartilage with the underlying bone. Poor lateral integration of the bone graft, as assessed on magnetic resonance imaging scan, and a low OAS were significantly associated with a poor Lysholm score and failure. A total of 3 patients had treatment failure, with 1 requiring total knee replacement at 5 years (Lysholm score of 33 at failure) and the other 2 requiring further surgical intervention because of persistent symptoms at 2 and 4 years, respectively (both had Lysholm score of 45 at failure). The Lysholm score in these patients before failure were still noted to be higher than at the preoperative level. Conclusion: The OsPlug technique shows significant improvement of functional outcome for up to 5 years in patients with high-grade osteochondritis dissecans or osteochondral defect. This is the first report describing association of bone graft integration with functional outcome after such a procedure. It also demonstrates histologic evidence of integration of the repair cartilage with the underlying bone graft.