Peter G. Bush

Chondrocyte death associated with human femoral osteochondral harvest as performed for mosaicplasty

BACKGROUND Autologous osteochondral transfer is an option for the treatment of articular defects. However, there are concerns about graft integration and the nature of the tissue forming the cartilage-cartilage bridge. Chondrocyte viability at graft and recipient edges is thought to be an important determinant of the quality of repair. The purpose of the present study was to evaluate early cell viability at the edges of osteochondral grafts from ex vivo human femoral condyles. METHODS Fresh human tissue was obtained from eleven knees at the time of total knee arthroplasty for the treatment of osteoarthritis. Osteochondral cylinders were harvested with use of a 4.5-mm-diameter mosaicplasty osteotome from regions of the anterolateral aspect of the femoral condyle that were macroscopically nondegenerate and histologically nonfibrillated. Plugs were assessed for marginal cell viability by means of confocal laser scanning microscopy. RESULTS The diameter of the cartilaginous portion of the osteochondral plugs was a mean (and standard error of the mean) of 4.84 +/- 0.12 mm (as determined on the basis of three plugs). This value was approximately 300 microm greater than the measured internal diameter of the osteotome. There was a substantial margin of superficial zone cell death (mean thickness, 382 +/- 68.2 microm), with >99% cell viability seen more centrally (as determined on the basis of five plugs). Demiplugs were created by splitting the mosaicplasty explants with a fresh number-11 scalpel blade. The margin of superficial zone cell death at the curved edge was significantly greater than that at the site of the scalpel cut (390.3 +/- 18.8 microm compared with 34.8 +/- 3.2 microm; p = 0.0286). Similar findings were observed when the cartilage alone was breached and the bone was left intact, with the margin of superficial zone cell death being significantly greater than that obtained in association with the straight scalpel incision (268 +/- 38.9 microm compared with 41.3 +/- 13.4 microm; p = 0.0286). The margin of superficial zone cell death showed no increase during the time-period between fifteen minutes and two hours after plug harvest. A mathematical approximation of the mosaicplasty region suggested that early cell death of this magnitude affects about one third of the superficial graft area. CONCLUSIONS The results of the present study suggest that mosaicplasty, while capable of transposing viable hyaline cartilage, is associated with an extensive margin of cell death that is likely to compromise lateral integration and articular reconstruction.

The volume and morphology of chondrocytes within non-degenerate and degenerate human articular cartilage

OBJECTIVE Cartilage swelling is an early event in osteoarthritis (OA). However, the response of chondrocytes to increased tissue hydration is unknown. This work studied the volume and morphology of living in situ human chondrocytes as a function of cartilage degeneration. METHODS The tibial plateaus from knee joints of 40 patients were obtained following above-knee amputations or knee arthroplasty, and degree of cartilage degeneration from 0 (non-eroded) to 3 (extensive fibrillations) was assessed using several criteria. In situ chondrocytes were labeled with fluorescent indicators (calcein for living cells, propidium iodide for dead cells) permitting the quantification of volume and visualisation of morphology of cells within the cartilage zones by confocal scanning laser microscopy (CLSM). RESULTS Chondrocyte volume within superficial and mid-zones, but not of deep zone cells, increased significantly (P<0.05 and P<0.02, respectively; one-way analysis of variance), with degree of cartilage hydration and degeneration. The volume increase ( approximately 90% for mid-zone chondrocytes, grade 3 cartilage) was greater than that which might occur following loss/excision of sub-chondral bone (<15% swelling). The CLSM technique utilised here revealed that approximately 40% of chondrocytes within all cartilage grades exhibited at least one cytoplasmic processes of <8 microm. The presence of these processes did not indicate a cell body of larger volume than cells without processes, and did not contribute to cell volume. CONCLUSIONS The volume of in situ chondrocytes within the superficial and mid-zones increased with cartilage degeneration. Cell swelling was greater than that expected from the increased hydration in OA, suggesting that an increase in chondrocyte volume might play a role in the changes to matrix metabolism occurring in OA.

Regulatory volume decrease (RVD) by isolated and in situ bovine articular chondrocytes

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently‐labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21°C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within ∼90 sec). Chondrocytes then exhibited rapid RVD (t1/2 ∼ 8 min), with cells from all zones returning to ∼3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell‐matrix interactions in mediating RVD.

Osmolarity influences chondrocyte death in wounded articular cartilage

BACKGROUND Mechanical injury results in chondrocyte death in articular cartilage. The purpose of the present study was to determine whether medium osmolarity affects chondrocyte death in injured articular cartilage. METHODS Osteochondral explants (n = 48) that had been harvested from the metacarpophalangeal joints of three-year-old cows were exposed to media with varying osmolarity (0 to 480 mOsm) for ninety seconds to allow in situ chondrocytes to respond to the altered osmotic environment. Explants were then wounded with a scalpel through the full thickness of articular cartilage, incubated in the same media for 2.5 hours, and transferred to 340-mOsm Dulbeccos Modified Eagle Medium (control medium) with further incubation for seven days. The spatial distribution of in situ chondrocyte death, percentage cell death, and marginal cell death at the wounded cartilage edge were compared as a function of osmolarity and time (2.5 hours compared with seven days) with use of confocal laser scanning microscopy. RESULTS In situ chondrocyte death was mainly localized to the superficial tangential zone of injured articular cartilage for the range of medium osmolarities (0 to 480 mOsm) at 2.5 hours and seven days. Therefore, a sample of articular cartilage from the superficial region (which included the scalpel-wounded cartilage edge) was studied with use of confocal laser scanning microscopy to compare the effects of osmolarity on percentage and marginal cell death in the superficial tangential zone. Compared with the control explants exposed to 340-mOsm Dulbeccos Modified Eagle Medium, percentage cell death in the superficial tangential zone was greatest for explants exposed to 0-mOsm (distilled water) and least for explants exposed to 480-mOsm Dulbeccos Modified Eagle Medium at 2.5 hours (13.0% at 340 mOsm [control], 35.5% at 0 mOsm, and 4.3% at 480 mOsm; p <or= 0.02 for paired comparisons) and seven days (9.9% at 340 mOsm [control], 37.7% at 0 mOsm, and 3.5% at 480 mOsm; p <or= 0.01 for paired comparisons). Marginal cell death in the superficial tangential zone decreased with increasing medium osmolarity at 2.5 hours (p = 0.001) and seven days (p = 0.002). There was no significant change in percentage cell death from 2.5 hours to seven days for explants initially exposed to any of the medium osmolarities. CONCLUSIONS Medium osmolarity significantly affects chondrocyte death in wounded articular cartilage. The greatest chondrocyte death occurs at 0 mOsm. Conversely, increased medium osmolarity (480 mOsm) is chondroprotective. The majority of cell death occurs within 2.5 hours, with no significant increase over seven days.

Passive osmotic properties of in situ human articular chondrocytes within non-degenerate and degenerate cartilage

Osteoarthritis is characterized by many factors, including proteoglycan loss, decreased collagen stiffness, and increased cartilage hydration. Chondrocyte swelling also occurs, and correlates with the degree of osteoarthritis, however, the cause is unknown but might be related to alterations to their passive osmotic properties. We have used two‐photon confocal laser scanning microscopy to measure the passive osmotic characteristics of in situ chondrocytes within relatively non‐degenerate and degenerate human tibial plateau cartilage, and in chondrocytes isolated from relatively non‐degenerate cartilage. Explants with bone attached were taken from a total of 42 patients undergoing arthroplasty and graded macroscopically and microscopically into two groups, grade 0 + 1 and grade 2 + 3. There was a significant increase in cartilage hydration between these two groups (P < 0.05), however, there was no change when medium osmolarity was varied over ∼0–480 mOsm. The passive osmotic behavior of in situ chondrocytes (at 4°C) was identical over a range of culture medium osmolarities (∼0–515 mOsm), however, the maximum swelling of cells within degenerate cartilage and isolated chondrocytes was greater compared to those in non‐degenerate cartilage. The swelling in the majority of in situ chondrocytes was accounted for by the reduced interstitial osmolarity occurring with cartilage degeneration. There was, however, a small population of in situ chondrocytes whose volume was in excess (≥2,500 μm3) of that predicted from the decreased interstitial osmotic pressure. These results show that for the majority of cells studied, the differences in passive chondrocyte volume between relatively non‐degenerate, degenerate, and isolated cells were entirely accounted for by changes to the extracellular osmolarity (180–515 mOsm).

Chondrocyte Death in Mechanically Injured Articular Cartilage―The Influence of Extracellular Calcium

Calcium is thought to be an important regulator of chondrocyte death associated with articular cartilage injury. Our objective was to determine the influence of extracellular calcium on chondrocyte death following mechanical injury. Using a surgically relevant model of sharp mechanical injury (with a scalpel) and confocal laser scanning microscopy (CLSM), in situ chondrocyte death was quantified within the full thickness of articular cartilage as a function of medium calcium concentration and time (2.5 h and 7 days). Exposure of articular cartilage to calcium‐free media (∼0 mM) significantly reduced superficial zone chondrocyte death after mechanical injury compared with exposure to calcium‐rich media (2–20 mM, ANOVA at 2.5 h, p = 0.002). In calcium‐rich media, although the extent of chondrocyte death increased with increasing medium calcium concentration, cell death remained localized to the superficial zone of articular cartilage over 7 days (ANOVA, p < 0.05). However, in calcium‐free media, there was an increase in chondrocyte death within deeper zones of articular cartilage over 7 days. The early (within hours) chondroprotective effect in calcium‐free media suggests that the use of joint irrigation solutions without added calcium may decrease chondrocyte death from mechanical injury during articular surgery. The delayed (within days) increase in chondrocyte death in calcium‐free media supports the use of calcium supplementation in media used during cartilage culture for tissue engineering or transplantation.

Functional analysis of mouse hepatocytes differing in DNA content: Volume, receptor expression, and effect of IFNγ

Polyploidy and binuclearity are characteristics of the mammalian liver. Increasing polyploidisation occurs with age and after administration of various drugs and chemicals. This study was designed to examine the function of ploidy by addressing several questions: (1) Does the increase in size of polyploid hepatocytes have any physiological function by altering surface receptor expression such as intercellular adhesion molecule‐1 (ICAM‐1, CD54) or IFNγR? and (2) Do polyploid cells respond differently to inflammatory cytokines such as interferon gamma (IFNγ)? We have developed a method to accurately measure the volume of live isolated hepatocytes using confocal microscopy and image analysis. Using flow cytometry, we have shown that the expression of ICAM‐1 increases with increasing DNA content and IFNγR is not detectable on isolated mouse hepatocytes. Diploid (2n), tetraploid (4n) and octoploid (8n) hepatocytes were found to be equally susceptible to IFNγ‐induced apoptosis in vitro. Although the function of polyploidy remains unanswered, we have described some of the characteristics of polyploidy in isolated hepatocytes and in vitro. J. Cell. Physiol. 191: 138–144, 2002.

Two-versus one photon excitation laser scanning microscopy: critical importance of excitation wavelength.

It is often anticipated that two-photon excitation (TPE) laser scanning microscopy should improve cell survival and tissue penetration relative to conventional one-photon excitation (OPE) confocal scanning laser microscopy (CLSM). However few studies have directly compared live cell imaging using one- vs two-photon laser scanning microscopy. We have used calcein-loaded in situ chondrocytes within cartilage as a model for quantitatively comparing these techniques. TPE reduced photo-bleaching and improved cell viability compared to OPE. Using improved detection sensitivity coupled with increased tissue penetration of the near infra-red TPE laser, it was possible to capture images deeper within the cartilage. However, the advantages of TPE vs OPE were strongly dependent on excitation wavelength. We conclude that optimising TPE conditions is essential for realizing the full benefits of this approach.

Abnormal human chondrocyte morphology is related to increased levels of cell‐associated IL‐1β and disruption to pericellular collagen type VI

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL‐1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2‐photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a “smooth” membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL‐1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno‐histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL‐1β‐associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r2 = 0.988) between the number of processes/cell (0–5) and IL‐1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL‐1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration.

New insights into function of the growth plate: clinical observations, chondrocyte enlargement and a possible role for membrane transporters.

The mammalian growth plate is a complex structure which is essential for the elongation of long bones. However, an understanding of how the growth plate functions at the cellular level is lacking. This review, summarises the factors involved in growth-plate regulation, its failure and the consequence of injury. We also describe some of the cellular mechanisms which underpin the increase in volume of the growth-plate chondrocyte which is the major determinant of the rate and extent of bone lengthening. We show how living in situ chondrocytes can be imaged using 2-photon laser scanning microscopy to provide a quantitative analysis of their volume. This approach should give better understanding of the cellular control of bone growth in both healthy and failed growth plates.