Arnold H. Schuurman

Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes

SummaryThe number of medical applications using autologous fibroblasts is increasing rapidly. We investigated thoroughly the procedure to isolate cells from skin using the enzymatic tissue dissociation procedure. Tissue digestion efficiency, cell viability, and yield were investigated in relation to size of tissue fragments, digestion volume to tissue ratio, digestion time, and importance of other protease activities present in Clostridium histolyticum collagenase (CHC) (neutral protease, clostripain, and trypsin). The results showed that digestion was optimal with small tissue fragments (2–3 mm3) and with volumes tissue ratios ≥2 ml/g tissue. For incubations ≤10 h, the digestion efficiency and cell isolation yields were significantly improved by increasing the collagenase, neutral protease, or clostripain activity, whereas trypsin activity had no effects. However, a too high proteolytic activity of one of the proteases present in CHC digestion solution or long exposure times interfered with cell viability and cell culture yields. The optimal range of CHC proteases activities per milliliter digestion solutions was determined for digestions ≤10 h (collagenase 2700–3900 Mandl U/ml, neutral protease 5100–10,000 caseinase U/ml, and clostripain 35–48 BAEE U/ml) and for longer digestions (>14 h) (collagenase 1350–3000 U/ml, neutral protease 2550–7700 U/ml, and clostripain 18–36 U/ml). Using these conditions, a maximum fibroblast expansion was achieved when isolated cells were seeded at 1×104 cells/cm2. These results did not only allow selection of optimal CHC batches able to digest dermal tissue with an high cell viability but also significantly increased the fibroblast yields, enabling us to produce autologous dermal tissue in a clinically acceptable time frame of 3 wk.

Keeping Osteocytes Alive: A Comparison of Drilling and Hammering K-Wires into Bone:

In this study, the insertion time and histological effects of drilling and hammering K-wires into bone are described. The insertion time was measured while drilling or hammering K-wires into the femurs and tibias of ten rabbits. Four K-wires, inserted into one hind limb, were used for histological examination directly after insertion and four K-wires inserted into the contralateral hind limb were used for the same measurements 4 weeks later. The specimens were scored for presence, or absence, of osteocytes, fragmentation of the bone edges, haemorrhage, microfractures, cortical reaction and callus formation around the pin track. The insertion time needed for drilling in K-wires was significantly longer than that of hammering. Drilling also resulted in the disappearance of the osteocytes in almost all sections while hammering did not have this effect but did result in more microfractures. Hammering K-wires may be a superior technique because it prevents osteonecrosis and requires a shorter insertion time.

Four-corner arthrodesis using the Quad Memory Staple

Range of motion, pain, consolidation and complications were evaluated for nine patients who underwent four-corner arthrodesis using the Quad Memory Staple (QMS) at a mean follow-up of 44 months. The mean pre-operative range of motion was 50° extension, 62° flexion, 9° radial deviation and 24° ulnar deviation. The postoperative range of motion was similar to previous studies at 32° extension, 31° flexion, 15° radial deviation and 20° ulnar deviation. The grip strength was 28 kg pre-operatively and 26 kg postoperatively. The mean pain score improved from 41 to 23 and the Disabilities of Arm, Shoulder, and Hand (DASH) score from 24 to 20. Non-union, haematoma and wound infection were not seen and eventually all four-corner fusions were consolidated. The main advantages of the QMS are its compressive property and the simple fixation technique. It gives good stability, enables early rehabilitation and avoids the risks of pin fixation methods.

The Value of 3.0-Tesla MRI in Diagnosing Scapholunate Ligament Injury

Objective To determine the sensitivity and specificity of 3.0-tesla (T) magnetic resonance imaging (MRI) and a dedicated hand coil in diagnosing scapholunate ligament (SLL) injury compared with intraoperative findings. Methods From January 2006 until September 2010, 3.0-T MRI scans were performed on 38 wrists (37 patients) with clinically unclear but suspected lesions of the SLL. These scans were evaluated by two experienced radiologists. Radiological findings were compared with intraoperative findings during arthrotomy. Sensitivity, specificity, accuracy, and positive and negative predictive value were calculated. Results An SLL lesion was identified during arthrotomy in 37 wrists. The first radiologist identified an SLL lesion on MRI in 26 wrists, all of which were confirmed intraoperatively. The second radiologist identified SLL lesions in 31 patients; however, intraoperatively it was found that there was no lesion of the ligament in one patient. Sensitivity ranged from 70 to 81% with a specificity of 100% and a positive predictive value of 97 to 100%. Accuracy measured 71 to 79%. Conclusions 3.0-T MRI of the wrist is moderately sensitive and very specific for detection of SLL lesions. However, if there is a high clinical suspicion of an SLL rupture, a 3.0-T MRI does not often have an additional value. Level of Evidence Diagnostic, level II.

Replantation versus Prosthetic Fitting in Traumatic Arm Amputations: A Systematic Review

Background Traumatic arm amputations can be treated with replantation or surgical formalization of the stump with or without subsequent prosthetic fitting. In the literature, many authors suggest the superiority of replantation. This systematic review compared available literature to analyze whether replantation is functionally and psychologically more profitable than formalization and prosthetic fitting in patients with traumatic arm amputation. Methods Functional outcome and satisfaction levels were recorded of patients with amputation levels below elbow, through elbow, and above elbow. Results Functional outcomes of 301 replantation patients and 172 prosthesis patients were obtained. In the replantation group, good or excellent functional scores were reported in 39% of above elbow, 55% of through elbow, and 50% of below elbow amputation cases. Nearly 100% of patients were satisfied with the replanted limb. In the prosthesis group, full use of the prosthesis was attained in 48% of above elbow and in 89% of below elbow amputation patients. Here, 29% of patients elected not to use the prosthesis for reasons including pain and functional superfluity. In both replantation patients and prosthesis wearers, a below elbow amputation yielded better functional results than higher amputation levels. Conclusions Replantation of a traumatically amputated arm leads to good function and higher satisfaction rates than a prosthesis, regardless of the objective functional outcome. Sensation and psychological well-being seem the two major advantages of replantation over a prosthesis. The current review of the available literature shows that in carefully selected cases replantation could be the preferred option of treatment.

A New Distal Radioulnar Joint Prosthesis

Pain and instability of the distal radioulnar joint (DRUJ) are common sequelae following a fracture of the distal radius. Many soft tissue procedures have been described, not all of which are successful. Ulnar head replacement prostheses are available but do not always provide stability. We designed a two-part, easy to implant, distal radioulnar prosthesis and implanted it in 19 patients. The first prototype was inserted in 2002 and is still in place. During the study, the design was changed twice, resulting in three groups with four patients in group A, five in group B, and ten in group C. Unfortunately all five prostheses in group B had to be removed because of loosening, while only two prostheses in group C had to be removed, for nonprosthetic reasons. For the 12 patients who retained their prosthesis, forearm function increased while grip strength increased significantly. Pain scores decreased and the Disabilities of the Arm, Shoulder, and Hand (DASH) score improved but remained high. We conclude that the prosthesis offers a new treatment option for ulnar instability following distal ulnar resection.

Inflammatory Mediators in Posttraumatic Radiocarpal Osteoarthritis

PURPOSE To identify the mediator profile in healthy, pre-osteoarthritis (OA) and end-stage OA radiocarpal joints. We hypothesized that there would be an increase in soluble mediators in posttraumatic wrist OA. METHODS We obtained radiocarpal synovial fluid samples from 3 groups of patients: healthy control (n = 12) samples were collected during wrist ganglion resection; pre-osteoarthritic (n = 16) samples, during a 3-ligament tenodesis procedure for complete scapholunate dissociation; and end-stage OA (n = 20) samples in patients with proven radiological OA changes. Using a multiplex enzyme-linked immunosorbent assay, we measured 12 mediators: interleukin (IL)-1β, tumor necrosis factor-α, oncostatin-M, interferon-γ, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-1RA, and osteoprotegerin. Statistical analysis was performed using analysis of variance and Bonferroni-corrected post hoc tests. RESULTS Mediators IL-6, IL-10, and interferon-γ were increased in OA wrists compared to healthy and pre-OA samples. Tumor necrosis factor-α, oncostatin-M, osteoprotegerin, IL-8, and IL-1RA were detected but not at increased levels in OA wrists. We found no differences between healthy and pre-OA joints in all 12 mediators. Mediators IL-4, IL-7, IL-13, and IL-1β were not detected in either healthy, pre-OA or end-stage OA samples. CONCLUSIONS We identified no differences between healthy and pre-OA samples, suggesting no alteration in inflammatory status at the time of the 3-ligament tenodesis procedure. Consequently, mechanical disturbance seems to be the driving force toward OA and OA-associated inflammation in this stage of scapholunate dissociation. Increased levels of interferon-γ, IL-6, and IL-10 confirm inflammatory changes in the mechanically disturbed posttraumatic radiocarpal joint.

Hammering K-Wires is Superior to Drilling with Irrigation

Cooling during drilling Kirschner wires is not always effective in preventing thermal related damage. In this study, we used a human in vitro model and compared temperature elevation, insertion time, and extraction force between three Kirschner wire insertion methods—drilling with and without irrigation and pneumatic hammering. Forty five Kirschner wires were inserted into 15 fresh human cadaver metacarpals. All three insertion methods were applied in each metacarpal. Drilling without irrigation resulted in a temperature elevation of 67.25 ± 5.4 ºC with significantly lower values for drilling with irrigation (4.15 ± 0.6 ºC) and pneumatic hammering (31.52 ± 3.4 ºC). The insertion time for pneumatic hammering (47.63 ± 8.8 s) was significantly lower compared to drilling without irrigation (263.16 ± 36.5 s) and drilling with irrigation (196.10 ± 28.5 s). Extraction forces after drilling without irrigation, drilling with irrigation, and pneumatic hammering were 39.85 ± 4.1 N, 57.81 ± 6.5 N, and 62.23 ± 6.7 N, respectively. Pneumatic hammering is superior to drilling without irrigation, especially when irrigation is not possible.

Functional Effects of the Adams Procedure: A Retrospective Intervention Study

Background Injury to the distal radioulnar joint (DRUJ) causes swelling and ulnar‐sided wrist pain. The Adams procedure stabilizes the DRUJ and shows promising short‐term results. Purpose We studied the long‐term functional outcome in patients who underwent the Adams procedure. We also tested the null hypothesis that there would be no difference in range of motion (ROM) and grip strength between the operated wrist compared to the unaffected side. Patients and Methods We identified 74 consecutive patients that were operated from March 2005 to February 2014. Twenty‐three patients responded to our invitation and underwent clinical follow‐up. Three patients had a re‐do of the Adams procedure after presenting with postoperative DRUJ instability, one was excluded due to additional wrist surgery not related to the initial procedure. Wrist functionality in the remaining 22 patients was examined by measuring ROM in all planes and grip strength of the operated and non‐operated hand. Functional outcome was measured by asking patients to complete the Disabilities of the Arm, Shoulder, and Hand (DASH) outcome measure and the Patient‐Rated Wrist and Hand Evaluation (PRWHE). Results After a mean follow‐up time of 5.0 years (standard deviation [SD] 2.0), we found that supination, pronation, and grip strength were significantly decreased in the operated wrist. Postoperative DASH and PRWHE‐scores averaged 13.1 (SD 18.6) and 20.3 (SD 15.1), respectively. Conclusion The Adams procedure affects, but largely restores pronation and supination, and clinical examination, DASH, and PRWHE scores indicate that the Adams procedure leaves patients with upper extremity functionality comparable to a healthy population. Level of Evidence Level IV.

A randomised placebo-controlled double-blind trial to assess the safety of intramuscular administration of allogeneic mesenchymal stromal cells for digital ulcers in systemic sclerosis: the MANUS Trial protocol

Introduction Systemic sclerosis (SSc) is an autoimmune disease characterised by inflammation, fibrosis and vasculopathy. Digital ulcers (DUs) are a frequent manifestation of vasculopathy in patients with SSc. Despite recent advances in pharmacological treatments, DU still have major health and economic implications. As there is currently no proven therapeutic strategy to promote DU healing, new treatments are urgently needed. Mesenchymal stem or stromal cells (MSCs) may provide a novel therapy for DU in SSc, because of their immunomodulatory and vasculoregenerative properties. Allogeneic MSC therapy involves functionally competent MSCs from healthy donors and may be used as ‘off-the-shelf’ available treatment. This study will evaluate whether allogeneic MSC therapy is a safe and potentially efficacious treatment for DU of SSc. Methods and analysis The MANUS (Mesenchymal stromal cells for Angiogenesis and Neovascularization in digital Ulcers of Systemic Sclerosis) Trial is a double-blind randomised placebo-controlled trial. 20 patients with SSc with refractory DU will be randomised to receive eight intramuscular injections with either placebo or 50*106 MSCs. The primary outcome is the toxicity of the treatment at 12 weeks after administration. Secondary outcomes include (serious) adverse events, number and time to healing of DU, pain, reported hand function, quality of life and SSc disease activity. We will also evaluate changes in nailfold capillaroscopy pattern, as well as biochemical parameters and biomarkers in peripheral blood and skin biopsies. Follow-up visits will be scheduled at 48 hours and 2, 4, 8, 12, 24 and 52 weeks post-treatment. If the results confirm safety, feasibility and potential efficacy, a large multicentre randomised controlled trial with longer follow-up will be initiated focusing on efficacy. Ethics and dissemination The study has been approved by the Dutch Central Committee on Research Concerning Human Subjects (protocol no: NL51705.000.15). The results will be disseminated through patient associations and conventional scientific channels. Trial registration number NCT03211793; Pre-results.