Morphological skin evaluation in the patients with systemic scleroderma before and after hematopoietic stem cell transplantation

Abstract

Some promising clinical results of hematopoietic stem cell transplantation (HSCT) are reported in severe autoimmune diseases. When treating the patients with systemic scleroderma (SSD), histological evaluation of skin fibrosis includes scoring of myofibroblasts that are associated with excessive deposition of extracellular matrix components. The aim of our study was to evaluate morphological condition of skin in SSD patients before and after transplantation of autologous hematopoietic bone marrow stem cells. Patients and methods Twenty-eight patients were observed at the National Research Medical Center (Nur-Sultan), at the age of 45+11 years old (2 males, 26 females) with verified diagnosis of SSD according to ACR/EULAR (2013). Duration of the disease was 12+5.4 years old. Control group (12 persons) received conventional basic therapy Treatment protocol for the main group included autologous HSCT. Resistance to immunosuppressive therapy was a pre-requisite for HSCT. Bone marrow aspiration was performed from the iliac crest, the autologous mononuclear cells were isolated in Percoll density gradient and incubated for 72 hours at 37°С. Autologous HSCT was performed at a mean dose of 88×106 cells in 200 mL of physiological saline i/v over 3 hours. Clinical effect was evaluated by the rec-ognized criteria (European Scleroderma Trials and Research Group, skin score by Rodnan). For morphologi-morphologi-cal studies, the punch biopsies of tibial skin were taken in 15 patients before therapy, and in 9 three months after the treatment. The cellular therapy was accompa-nied by improved skin condition. The paraffin sections were stained with hematoxilin and eosin, as well as by Masson-trichrome technique. For electron microscopy, the skin biopsies were processed by conventional meth-od, then being Epon-embedded. Semi-thin slices were stained with Methylene Blue, Azur II and basic fuchsine. For EM, the ultrathin sections were contrasted with ura-nyl acetate and lead citrate. Results Skin of SSD patients before treatment was characterized by induration and dystrophy and epithelial destruction, sclerosis and hyalinosis of dermal connective tissue, pa-thology of microcirculatory vessels. Ultrastructure of myofibroblasts in the sclerotized derma was characterized by functional overload. The active participants of fibrillogenesis were located in perivascular area, being represented by lymphocytes and fibroblasts of a specific SSD-specific population producing higher amounts of collagen and interstitial matrix. However, three months after HSCT, the main group of the patients exhibited a pronounced clinical effect with sufficient decrease of skin induration, reduced dysphagia, mitigation of mus-cle contractures, couping vasospasm attacks (Raynaud syndrome). Skin density was significantly decreased, with Rodnan scores changed from 12.9 to 8.7 (only 1-point decrease in the controls). HSCT promoted bi-odegradation of skin fibrotic tissue in SSD patients. The cal myofibroblasts were subjected to destruction, multiple and prolonged capillaries were observed, the cell composition of perivascular infiltrate shifted to normal state. Numerous phagocytic and secretory macrophages ap-peared, thus suggesting angiogenesis induction, tissue remodeling, regulation of fibroclast population, sup-pression of T-and B-lymphocytes playing an important role on SSD pathogenesis. Extensive telocyte connec-tions presumed their participation in neoangiogenesis and transmission of regeneration signaling. Conclusion Transplantation of cultured autologous hematopoietic marrow stem cells in SSD patients promoted biodegra-dation of sclerotized dermal layer, as well as angiogen-esis stimulation, restoration of epithelium and skin ap-pendages 3 months after HSCT, thus corresponding to improvement of clinical symptoms.