Johan Thorfinn

Flexor Tendon Tissue Engineering: Acellularization of Human Flexor Tendons with Preservation of Biomechanical Properties and Biocompatibility

OBJECTIVE Acellular human tendons are a candidate scaffold for tissue engineering flexor tendons of the hand. This study compared acellularization methods and their compatibility with allogeneic human cells. METHOD Human flexor tendons were pretreated with 0.1% ethylenediaminetetracetic acid (EDTA) for 4  h followed by 24  h treatments of 1% Triton X-100, 1% tri(n-butyl)phosphate, or 0.1% or 1% sodium dodecyl sulfate (SDS) in 0.1% EDTA. Outcomes were assessed histologically by hematoxylin and eosin and SYTO green fluorescent nucleic acid stains and biochemically by a QIAGEN DNeasy kit, Sircol collagen assay, and 1,9 dimethylmethylene blue glycosaminoglycan assay. Mechanical data were collected using a Materials Testing System to pull to failure tendons acellularized with 0.1% SDS. Acellularized tendons were re-seeded in a suspension of human dermal fibroblasts. Attachment of viable cells to acellularized tendon was assessed biochemically by a cell viability assay and histologically by a live/dead stain. Data are reported as mean±standard deviation. RESULT Compared with the DNA content of fresh tendons (551±212  ng DNA/mg tendon), only SDS treatments significantly decreased DNA content (1% SDS [202.8±37.4  ng DNA/mg dry weight tendon]; 0.1% SDS [189±104  ng DNA/mg tendon]). These findings were confirmed by histology. There was no decrease in glycosaminoglycans or collagen following acellularization with SDS. There was no difference in the ultimate tensile stress (55.3±19.2 [fresh] vs. 51.5±6.9 [0.1% SDS] MPa). Re-seeded tendons demonstrated attachment of viable cells to the tendon surface using a viability assay and histology. CONCLUSION Human flexor tendons were acellularized with 0.1% SDS in 0.1% EDTA for 24  h with preservation of mechanical properties. Preservation of collagen and glycoaminoglycans and re-seeding with human cells suggest that this scaffold is biocompatible. This will provide a promising scaffold for future human flexor tendon tissue engineering studies to further assess biocompatibility through cell proliferation and in vivo studies.

Sitting pressure and perfusion of buttock skin in paraplegic and tetraplegic patients, and in healthy subjects : a comparative study

The distribution of sitting pressure and ability to respond with reactive hyperaemia were studied in a group of paraplegic and tetraplegic patients ( n = 8) with spinal cord lesions and healthy controls ( n = 10) using a pressure sensitive plate and laser Doppler perfusion imager. The results show that the mean sitting pressure of the patients was 9.9 N/cm 2 (left) and 11.7 N/cm 2 (right) compared with 3.5 N/cm 2 controls. The differences were significant on both the left ( p < 0.01) and right ( p < 0.05) sides. The maximum pressure in patients was 42.9 N/cm 2 (left) and 48.7 N/cm 2 (right), and in controls ( p < 0.01). Both groups showed a reduction in skin perfusion in the seat area during sitting compared with unloaded resting, and in the controls it was significantly increased ( p < 0.001 on both sides) during the reactive hyperaemic phase immediately after sitting. Compared with the preload values, the patients showed a similar but slightly weaker picture significant on the right side ( p < 0.05), but not on the left. The hyperaemia was not uniformly distributed, but occurred where the pressure was greater than 2 N/cm 2 . There was no correlation between the amount of reactive hyperaemia and absolute values of sitting pressures. We conclude that tetraplegic and paraplegic patients have significantly higher sitting pressures than normal controls, and that the hyperaemic response in the buttock region in the upright position after pressure load is slightly weaker in the patients, which could be of importance for the development of decubitus ulcers.

Tissue Engineering of Flexor Tendons: The Effect of a Tissue Bioreactor on Adipoderived Stem Cell-Seeded and Fibroblast-Seeded Tendon Constructs

PURPOSE Tissue-engineered flexor tendons could eventually be used for reconstruction of large tendon defects. The goal of this project was to examine the effect of a tissue bioreactor on the biomechanical properties of tendon constructs seeded with adipoderived stem cells (ASCs) and fibroblasts (Fs). METHODS Rabbit rear paw flexor tendons were acellularized and seeded with ASCs or Fs. A custom bioreactor applied a cyclic mechanical load of 1.25 N at 1 cycle/minute for 5 days onto the tendon constructs. Three additional groups were used as controls: fresh tendons and tendons reseeded with either ASCs or Fs that were not exposed to the bioreactor treatment and were left in stationary incubation for 5 days. We compared the ultimate tensile stress (UTS) and elastic modulus (EM) of bioreactor-treated tendons with the unloaded control tendons and fresh tendons. Comparison across groups was assessed using one-way analysis of variance with the significance level set at p<.05. Pairwise comparison between the samples was determined by using the Tukey test. RESULTS The UTS and EM values of bioreactor-treated tendons that were exposed to cyclic load were significantly higher than those of unloaded control tendons. Acellularized tendon constructs that were reseeded with ASCs and exposed to a cyclic load had a UTS of 66.76 MPa and an EM of 906.68 MPa; their unloaded equivalents had a UTS of 47.90 MPa and an EM of 715.57 MPa. Similar trends were found in the fibroblast-seeded tendon constructs that were exposed to the bioreactor treatment. The bioreactor-treated tendons approached the UTS and EM values of fresh tendons. Histologically, we found that cells reoriented themselves parallel to the direction of strain in response to cyclic strain. CONCLUSIONS The application of cyclic strain on seeded tendon constructs that were treated with the bioreactor helped achieve a UTS and an EM comparable with those of fresh tendons. Bioreactor pretreatment and alternative cell lines, such as ASCs and Fs, might therefore contribute to the in vitro production of strong tendon material.

Flexor Tendon Tissue Engineering: Temporal Distribution of Donor Tenocytes versus Recipient Cells

Background: Tissue-engineered tendon material may address tendon shortages in mutilating hand injuries. Tenocytes from rabbit flexor tendon can be successfully seeded onto acellularized tendons that are used as tendon constructs. These constructs in vivo exhibit a population of tenocyte-like cells; however, it is not known to what extent these cells are of donor or recipient origin. Furthermore, the temporal distribution is also not known. Methods: Tenocytes from New Zealand male rabbits were cultured and seeded onto acellularized rabbit forepaw flexor tendons (n = 48). These tendon constructs were transplanted into female recipients. Tendons were examined after 3, 6, 12, and 30 weeks using fluorescent in situ hybridization to detect the Y chromosome in the male donor cells. One unseeded, acellularized allograft in each animal was used as a control. Results: The donor male tenocytes populate the epitenon and endotenon of the grafts at greater numbers than the recipient female tenocytes at 3 and 6 weeks. The donor and recipient tenocytes are present jointly in the grafts until 12 weeks. At 30 weeks, nearly all cells are recipient tenocyte-like cells. Conclusions: Donor male cells survive in decreasing numbers over time until 30 weeks. The presence of cells in tissue-engineered tendon grafts has been shown in prior studies to add to the strength of the constructs in vitro. This study shows that recipient cells can migrate into and repopulate the tendon construct. Cell seeding onto tendon material may create stronger constructs that will allow the initiation of motion earlier.

Bioreactor optimization of tissue engineered rabbit flexor tendons in vivo

Tissue-engineered rabbit flexor tendons reseeded with cells are stronger in vitro after culture in a bioreactor. It is not known whether this effect persists in vivo. Tenocytes from New Zealand white rabbits were seeded onto rabbit rear paw flexor tendons that were deprived of cells and exposed to cyclic strain in a bioreactor. Reseeded constructs that were kept unloaded in a medium for 5 days were used as controls. The tendons were implanted to bridge a zone II defect in the rabbit. After explantation 4 weeks later, the ultimate tensile strength (UTS) and elastic modulus (EM) were determined. Tendon constructs that were exposed to cyclic strain had significantly improved UTS and EM. Histology showed that cellularity was increased in the bioreactor tendons.

Perfusion of the skin of the buttocks in paraplegic and tetraplegic patients, and in healthy subjects after a short and long load

In patients with spinal cord injuries (n=8) and healthy controls (n=8) the hyperaemic response in the buttock skin after sitting on a hard surface was studied using a laser Doppler perfusion imager. They sat for three minutes (short load), or 15 minutes (long load). An exponential mathematical function was used to compare the mean perfusion during the observed interval. The results showed that preloading perfusion is significantly higher among patients than healthy subjects. In both groups, the microcirculation of the skin increased significantly after loading, and peak perfusion was significantly lower after the short load. The mean perfusion was higher among the patients after both loadings, which suggests that there was stronger ischaemic provocation. The main outcome was that there was a dose-response relation between duration of loading and intensity of reactive hyperaemia, and that patients with spinal cord injuries have greater perfusion before and after loading than healthy controls.

Hyperaemic changes in forearm skin perfusion and RBC concentration after increasing occlusion times

Tissue occlusion and the hyperaemic response upon reperfusion can be used as a tool to assess microvascular function in various vascular diseases. Currently, laser Doppler flowmetry (LDF) is applied most often to measure hyperaemic responses. In this study, we have applied tissue viability imaging (TiVi) and LDF to measure the change in red blood cell concentration and perfusion in the skin after occlusions of the forearm with increasing duration. We have found that there is a strong correlation between the changes in perfusion and red blood cell (RBC) concentration during post-occlusive hyperaemia (perfusion: r=0.80; RBC concentration: r=0.94). This correlation increases with longer occlusion durations (1, 5 and 10min). Furthermore, for both perfusion and RBC concentration, the maximum responses (perfusion: r(2)=0.59; RBC concentration: r(2)=0.78) and the recovery times (perfusion: r(2)=0.62; RBC concentration: r(2)=0.91) increase linearly with the duration of the occlusion. Maximum responses and recovery times were more reproducible for RBC concentration (as measured with TiVi) than for perfusion (as measured with LDF). These results show that perfusion and RBC concentration are related during post-occlusive hyperaemia and that TiVi can be used as a tool in the assessment of hyperaemic responses that has advantages in terms of reproducibility, sensitivity and ease of use.

Perfusion of buttock skin in healthy volunteers after long and short repetitive loading evaluated by laser Doppler perfusion imager

Frequent unloading is vital to avoid pressure ulcers of the seat area in patients with injuries to the spinal cord. The duration of unloading is probably as important as that of the sitting period in the prophylaxis of pressure ulcers. The aim of this study was to investigate the microcirculatory reactions after occlusion of the buttock skin after repeated ischaemic provocation. The perfusion of buttock skin was studied with a laser Doppler perfusion imager (LDPI) in healthy people after short and long periods of sitting (repeated four times). The perfusion increased significantly during the consecutive loadings compared with the first loading, and this effect was more profound after the long load. Repeated periods of ischaemia of the buttock skin without allowing the tissues to recover resulted in increasing reactive hyperaemia, and are therefore probably more damaging than single loadings. This is important when establishing clinical guidelines for the prophylaxis of pressure ulcers in patients with spinal cord injuries.

Laser Doppler perfusion imaging of the radial forearm flap: a clinical study.

Laser Doppler perfusion imaging (LDPI) allows non-invasive assessment of blood flow in a predefined area of skin rather than at one single point. We have used LDPI to study the pattern of skin blood flow in the radial forearm flap before and after the flap has been raised. The data were collected from a consecutive series of 11 patients with cancer of the oral cavity or oropharynx in whom the radial forearm flap had been used during the reconstructive procedure. Reperfusion leads to an immediate hyperaemic response both in the flap and the surrounding skin. This hyperaemia remains for at least the first 30 minutes after reperfusion. The perfusion of the radial side of the forearm skin distal to the flap is significantly lower than that on the ulnar side after the skin island has been raised and the distal artery divided. We suggest that LDPI is useful for monitoring the perfusion of free skin flaps.

Technological changes in the management of prostate cancer result in increased healthcare costs: A retrospective study in a defined Swedish population

OBJECTIVE In two previous studies we calculated direct costs for men with prostate cancer who died in 1984-85 and 1992-93, respectively. We have now performed a third cost analysis to enable a longitudinal cost comparison. The aim was to calculate direct costs for the management of prostate cancer, describe the economic consequences of technological changes over time and estimate total direct costs for prostate cancer in Sweden. MATERIAL AND METHODS A total of 204 men in a defined population with a diagnosis of prostate cancer and who died in 1997-98 were included. Data on utilization of health services were extracted from clinical records from time of diagnosis to death from a university hospital and from one county hospital in the county of Ostergötland. RESULTS The average direct cost per patient has been nearly stable over time (1984-85: 143 000 SEK; 1992-93: 150 000 SEK; 1997-98: 146 000 SEK). The share of costs for drugs increased from 7% in 1992-93 to 17% in 1997-98. The total direct costs for prostate cancer in Sweden have increased over time (1994-85: 610 MSEK; 1992-93: 860 MSEK; 1997-98: 970 MSEK). CONCLUSIONS Two-thirds of the total cost is incurred by inpatient care. The share of the total costs for drugs is increasing due to increased use of gonadotrophin-releasing hormone analogues. Small changes in average direct costs per patient despite greater use of technology are explained by the fact that more prostate cancers are detected at the early stages.