Simon Farnebo

Human flexor tendon tissue engineering: in vivo effects of stem cell reseeding.

Background: Tissue-engineered human flexor tendons may be an option to aid in reconstruction of complex upper extremity injuries with significant tendon loss. The authors hypothesize that human adipose-derived stem cells remain viable following reseeding on human tendon scaffolds in vivo and aid in graft integration. Methods: Decellularized human flexor tendons harvested from fresh-frozen cadavers and reseeded with green fluorescent protein–labeled pooled human adipose-derived stem cells were examined with bioluminescent imaging and immunohistochemistry. Reseeded repaired tendons were compared biomechanically with unseeded controls following implantation in athymic rats at 2 and 4 weeks. The ratio of collagen I to collagen III at the repair site was examined using Sirius red staining. To confirm cell migration, reseeded and unseeded tendons were placed either in contact or with a 1-mm gap for 12 days. Green fluorescent protein signal was then detected. Results: Following reseeding, viable cells were visualized at 12 days in vitro and 4 weeks in vivo. Biomechanical testing revealed no significant difference in ultimate load to failure and 2-mm gap force. Histologic evaluation showed host cell invasion and proliferation of the repair sites. No increase in collagen III was noted in reseeded constructs. Cell migration was confirmed from reseeded constructs to unseeded tendon scaffolds with tendon contact. Conclusions: Human adipose-derived stem cells reseeded onto decellularized allograft scaffolds are viable over 4 weeks in vivo. The movement of host cells into the scaffold and movement of adipose-derived stem cells along and into the scaffold suggests biointegration of the allograft.

Augmentation of tendon healing with an injectable tendon hydrogel in a rat Achilles tendon model.

Background: Many unsolved problems in plastic and hand surgery are related to poor healing of acute and chronic tendon injuries. The authors hypothesized that tendon healing could be augmented by the addition of a tendon-derived, extracellular matrix hydrogel that would guide tissue regeneration. Methods: Both Achilles tendons of 36 Wistar rats were given full-thickness injuries approximately 5 mm long and 0.5 mm wide from the tendon insertion at the calcaneus to the midsubstance. The hydrogel was injected into the injury site of one leg and compared with control saline in the other. The ultimate failure load, ultimate tensile stress, and stiffness were evaluated at 2, 4, and 8 weeks. Tendon cross-sections underwent histologic analysis (hematoxylin and eosin and picrosirius red) after the animals were killed. Statistical analysis of biomechanical data was performed using a paired t test. Results: There was no significant difference in strength between gel and saline injections in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.42), or stiffness (p = 0.76) at 2 weeks. However, there was a significant difference in ultimate failure load (74.8 ± 11.6 N versus 58.4 ± 14.2 N; p = 0.02) at 4 weeks. The difference in ultimate tensile stress (p = 0.63) and stiffness (p = 0.08) remained insignificant. By 8 weeks, there was no significant difference in strength in ultimate failure load (p = 0.15), ultimate tensile stress (p = 0.39), or stiffness (p = 0.75). Conclusions: Treatment with the tendon hydrogel significantly increases the ultimate failure load of tendons at the critical 4-week time point, and is a promising method for augmentation of tendon healing.

Assessment of blood flow changes in human skin by microdialysis urea clearance.

Please cite this paper as: Farnebo, Zettersten, Samuelsson, Tesselaar and Sjöberg (2011). Assessment of Flood Flow Changes in Human Skin by Microdialysis Urea Clearance. Microcirculation 18(3), 198–204.

Urea clearance: a new method to register local changes in blood flow in rat skeletal muscle based on microdialysis.

Increasing evidence suggests that local blood flow should be monitored during microdialysis (MD) as the recovery of analytes is affected by local blood flow. At present ethanol clearance is the standard technique for this purpose, but it is not functional at very low perfusion velocities. Here, we introduce a technique for MD whereby local tissue blood flow is recorded by the use of urea clearance (changes inflow/outflow concentration), in conjunction with measurements of tissue metabolism (glucose, lactate and puruvate). MD probes were inserted into the gracilis muscle of 15 rats and perfused with a medium containing urea (20 mmol l−1). Changes in muscle blood flow were made by addition of noradrenaline (5 μg ml−1) to the perfusion medium at two perfusion velocities (0·6 and 0·4 μl min−1). The clearance of urea from the perfusion medium was then calculated and examined in relation to the dose of noradrenaline and to the coexisting changes in extracellular metabolites. The results showed reproducible and dose‐dependent changes in blood flow that were induced by noradrenaline. These were characterized by dose‐dependent changes in the urea clearance as well as blood‐flow‐specific changes in the MD metabolic markers (reduction in glucose and increase in lactate). The sensitivity for blood flow changes as assessed by urea clearance (MD) was increased at 0·4 compared with the 0·6 μl min−1 perfusion speed. The results indicate that inclusion of urea to the perfusion medium may be used to monitor changes in skeletal muscle blood flow at low perfusion velocities and in parallel assess metabolic variables with a high recovery (>90%).

Non-Invasive Measurement of Skin Microvascular Response during Pharmacological and Physiological Provocations

Introduction Microvascular changes in the skin due to pharmacological and physiological provocations can be used as a marker for vascular function. While laser Doppler flowmetry (LDF) has been used extensively for measurement of skin microvascular responses, Laser Speckle Contrast Imaging (LSCI) and Tissue Viability Imaging (TiVi) are novel imaging techniques. TiVi measures red blood cell concentration, while LDF and LSCI measure perfusion. Therefore, the aim of this study was to compare responses to provocations in the skin using these different techniques. Method Changes in skin microcirculation were measured in healthy subjects during (1) iontophoresis of sodium nitroprusside (SNP) and noradrenaline (NA), (2) local heating and (3) post-occlusive reactive hyperemia (PORH) using LDF, LSCI and TiVi. Results Iontophoresis of SNP increased perfusion (LSCI: baseline 40.9±6.2 PU; 10-min 100±25 PU; p<0.001) and RBC concentration (TiVi: baseline 119±18; 10-min 150±41 AU; p = 0.011). No change in perfusion (LSCI) was observed after iontophoresis of NA (baseline 38.0±4.4 PU; 10-min 38.9±5.0 PU; p = 0.64), while RBC concentration decreased (TiVi: baseline 59.6±11.8 AU; 10-min 54.4±13.3 AU; p = 0.021). Local heating increased perfusion (LDF: baseline 8.8±3.6 PU; max 112±55 PU; p<0.001, LSCI: baseline 50.8±8.0 PU; max 151±22 PU; p<0.001) and RBC concentration (TiVi: baseline 49.2±32.9 AU; max 99.3±28.3 AU; p<0.001). After 5 minutes of forearm occlusion with prior exsanguination, a decrease was seen in perfusion (LDF: p = 0.027; LSCI: p<0.001) and in RBC concentration (p = 0.045). Only LSCI showed a significant decrease in perfusion after 5 minutes of occlusion without prior exsanguination (p<0.001). Coefficients of variation were lower for LSCI and TiVi compared to LDF for most responses. Conclusion LSCI is more sensitive than TiVi for measuring microvascular changes during SNP-induced vasodilatation and forearm occlusion. TiVi is more sensitive to noradrenaline-induced vasoconstriction. LSCI and TiVi show lower inter-subject variability than LDF. These findings are important to consider when choosing measurement techniques for studying skin microvascular responses.

Physicochemical decellularization of composite flexor tendon-bone interface grafts.

Background: Extremity injuries involving tendon attachment to bone are difficult to address. Clinically, tendon-bone interface allografts must be decellularized to reduce immunogenicity. Composite grafts are difficult to decellularize because chemical agents cannot reach cells between tissues. In this study, the authors attempted to optimize tendon-bone interface graft decellularization. Methods: Human flexor digitorum profundus tendons with attached distal phalanx were harvested from cadavers and divided into four groups. Group 1 (control) was untreated. Group 2 (chemical) was chemically treated with 5% peracetic acid, 0.1% ethylenediaminetetraacetic acid, and 0.1% sodium dodecyl sulfate. Group 3 (low-power) underwent targeted ultrasonication for 3 minutes (22,274 J, 126W) followed by chemical decellularization. Group 4 (high-power) underwent targeted ultrasonication for 10 minutes (88,490 J, 155W) followed by chemical decellularization. Decellularization was assessed histologically with hematoxylin and eosin stain and stains for major histocompatibility complex I stains. Cell counts were performed. The ultimate tensile load of decellularized grafts (group 4) were compared with pair-matched untreated grafts (group 1). Results: Average cell counts were 100 ± 41, 27 ± 10, 12 ± 11, and 6 ± 11 per high-power field for groups 1, 2, 3, and 4, respectively (p < 0.001). Decellularization using physical and chemical treatments (groups 3 and 4) resulted in substantial reduction of cells and major histocompatibility complex I molecules. There was no difference in ultimate tensile load between treated (group4) and untreated (group 1) samples (p > 0.5). Conclusions: Physicochemical decellularization of tendon-bone interface grafts using targeted ultrasonication and chemical treatment resulted in near-complete reduction in cellularity and maintenance of tensile strength. In the future, these decellularized composite scaffolds may be used for reconstruction of tendon-bone injuries.

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.

Decellularized tendon-bone composite grafts for extremity reconstruction: an experimental study.

Background: Restoration of biomechanical strength following surgical reconstruction of tendon or ligament insertion tears is challenging because these injuries typically heal as fibrous scars. The authors hypothesize that injuries at the tendon-bone interface would benefit from reconstruction with decellularized composite tendon-bone grafts. Methods: Tendon-bone grafts were harvested from Sprague-Dawley rats. Grafts subjected to decellularization were compared histologically and biomechanically with untreated grafts ex vivo and in a new in vivo model. Wistar rats underwent Sprague-Dawley allograft reconstruction using a pair-matched design. The rats were killed at 2 or 4 weeks. B-cell and macrophage infiltration was determined using immunohistochemistry, and explants were tested biomechanically. Results: Decellularization resulted in a decrease in cells from 164 ± 61 (untreated graft) to 13 ± 7 cells per high-power field cells (p < 0.005) and a corresponding significant decrease in DNA content, and preserved scaffold architecture of the tendon-bone interface. Biomechanical comparison revealed no difference in failure load (p = 0.32), ultimate tensile stress (p = 0.76), or stiffness (p = 0.22) between decellularized grafts and untreated controls. Following in vivo reconstruction with tendon-bone interface grafts, decellularized grafts were stronger than untreated grafts at 2 weeks (p = 0.047) and at 4 weeks (p < 0.005). A persistent increase in B-cell and macrophage infiltration was observed in both the capsule surrounding the tendon-bone interface and the tendon substance in untreated controls. Conclusion: Decellularized tendon-bone grafts display better biomechanical properties at early healing time points and a decreased immune response compared with untreated grafts in vivo.

Reconstruction of the Tendon–Bone Insertion With Decellularized Tendon–Bone Composite Grafts: Comparison With Conventional Repair

PURPOSE Injuries involving the tendon-bone interface (TBI) are difficult to address. Standard techniques typically lead to diminished strength of the healed insertion site. We hypothesized that these injuries would benefit from being reconstructed with decellularized composite grafts replacing both tendon and bone. To test this hypothesis, decellularized grafts were compared with conventional pullout repairs in an in vivo animal model. METHODS We harvested 48 Achilles TBI grafts from rats and decellularized them. Tendon-bone interface graft reconstruction and pullout repairs were compared using a pair-matched design. Biomechanical properties were evaluated at 2, 4, 8, and 12 weeks. We evaluated histological analysis of insertion morphology and collagen type I/III content. RESULTS There was a significant increase in ultimate failure load (35 ± 11 vs 24 ± 7 N) and ultimate tensile stress (1.5 ± 0.3 vs 1.0 ± 0.4 N/mm(2)) of the TBI grafts compared with pullout repairs at 2 weeks. These differences remained at 4 weeks. At 12 weeks, both TBI grafts and pullout repairs were as strong as native tissue and not significantly different from each other. Histology showed a more organized extracellular matrix in the TBI graft group at the early time points. Repopulation of the decellularized grafts increased over time. At 12 weeks, the insertion points of both groups were richly populated with cells that possessed morphologies similar to those found in native TBI. CONCLUSIONS This study showed that decellularized TBI grafts were stronger compared with conventional pullout repairs at 2 and 4 weeks but were comparable at 12 weeks. A more organized extracellular matrix and different collagen composition in the early time points may explain the observed differences in strength. CLINICAL RELEVANCE In the future, decellularized TBI grafts may be used to reconstruct tendon-bone insertion tears in multiple areas including the flexor tendon system.

Microvascular blood flow in scalds in children and its relation to duration of wound healing: A study using laser speckle contrast imaging

BACKGROUND Microvascular perfusion changes in scalds in children during the first weeks after injury is related to the outcome of healing, and measurements of perfusion, based on laser Doppler imaging, have been used successfully to predict the need for excision and grafting. However, the day-to-day changes in perfusion during the first weeks after injury have not to our knowledge been studied in detail. The aim of this study, based on a conservative treatment model where excision and grafting decisions were delayed to day 14 after injury, was to measure changes in perfusion in scalds using laser speckle contrast imaging (LSCI) during the first three weeks after injury. METHODS We measured perfusion with LSCI in 34 patients at regular intervals between 6h after injury until complete reepithelialization or surgery. Duration of healing was defined as the time to complete reepithelialization. RESULTS Less perfusion, between 6 and 96h after injury, was associated with longer duration of healing with the strongest association occurring between 72 and 96h. Burns that healed within 14 days had relatively high initial perfusion, followed by a peak and subsequent slow decrease. Both the maximum perfusion and the time-to-peak were dependent on the severity of the burn. Burns that needed excision and grafting had less initial perfusion and a gradual reduction over time. CONCLUSION The perfusion in scalds in children shows characteristic patterns during the first weeks after injury depending on the duration of wound healing, the greatest difference between wounds of different severity being on the 4th day. Perfusion should therefore preferably be measured on the fourth day if it is to be used in the assessment of burn depth.