Application of a three-dimensional graft of autologous osteodifferentiated adipose stem cells in patients undergoing minimally invasive transforaminal lumbar interbody fusion: clinical proof of concept.

BACKGROUND: The authors applied a scaffold-free osteogenic three-dimensional (3D) graft made of adipose-derived mesenchymal stem cells (AMSCs) in patients undergoing minimally invasive transforaminal lumbar interbody fusion (MI-TLIF). METHODS: Three patients (two patients and one patient with 1 and 2 levels, respectively) with degenerative spondylolisthesis underwent MI-TLIF with 3D graft made of AMSCs. To obtain the AMSCs, fatty tissue was collected from the abdomen by lipoaspiration and differentiated afterwards in our Cell/Tissue bank. Clinical outcomes, including the Oswestry Disability Index (ODI) and visual analog scale (VAS) as well as fusion status were assessed preoperatively and up to 12 months postoperatively. RESULTS: At 12 months, all four operated AMSC levels could be assessed (n = 4). Grade 3 fusion could be confirmed at two levels out of four. Mean VAS score improved from 8.3 to 2 and ODI also improved from 47 to 31%. No donor site complication was observed. The final AMSC osteogenic product was stable, did not rupture with forceps manipulation, and was easily implanted directly into the cage with no marked modification of operating time. CONCLUSIONS: A scaffold-free 3D graft made of AMSCs can be manufactured and used as a promising alternative for spinal fusion procedures. Nevertheless, further studies of a larger series of patients are needed to confirm its effectiveness.

The fate and role of bone graft-derived cells after autologous tendon and bone transplantation into the bone tunnel.

BACKGROUND: Grafting bone between the tendon graft and the bone tunnel in anterior cruciate ligament reconstruction increases the mechanical strength of the tendon graft. However, the biological role of the bone graft is unclear. The purpose of this research was to elucidate the role of bone graft cells after autologous tendon graft into the bone tunnel with an autologous bone graft in green fluorescent protein (GFP) transgenic rats. METHODS: The Achilles tendons of Sprague-Dawley (SD) wild-type rats and bone of GFP rats were harvested and transplanted into bone tunnels drilled in the femurs at the knees of SD rats. The femurs were harvested at 1, 2, and 4 weeks after transplantation and histologically investigated using hematoxylin and eosin staining and immunostaining of heat shock protein 47 (HSP47), macrophages, and type I and type III collagens. Biomechanical tests were performed on the tendon graft 2 and 4 weeks after transplantation to evaluate the ultimate force to failure. RESULTS: A small number of GFP-positive cells was seen in the tendon graft 2 weeks after transplantation. The cell count in the tendon graft was increased at 4 weeks after transplantation. HSP47-positive cells and macrophage-stained cells present in the tendon graft corresponded with the GFP-positive cells. By 2 weeks after transplantation, the relative areas of immunostained type I and III collagens in the tendon graft had declined significantly in the bone graft group compared to the control. The ultimate failure load in the bone graft group was higher than that in the control group at both 2 and 4 weeks after transplantation. CONCLUSIONS: This research showed that, within 4 weeks of transplantation, bone graft cells migrate to the tendon graft, where they differentiate into cells involved in collagen production and macrophages. Bone graft cells may contribute to the early stage remodeling of tendon grafts.

Polytherapy in bone regeneration: clinical applications and preliminary considerations.

Polytherapy, namely the simultaneous application of three fundamental elements necessary for bone regeneration (growth factors, osteogenic cells and osteoconductive scaffolds) seems to lead to a very high success rate in the treatment of complex non-union (NU) cases and critical bone defects. NU are reported in 5-10% of long bone fractures. The use of autologous bone grafts has been long-considered the gold standard for the treatment of these cases. However the harvesting procedure from the iliac crest increases surgery time and presents some donor site complications which may be elevated. In recent years, surgeons have some alternatives to autologous grafting such as: application of organic or synthetic bone substitute, application of mesenchymal stromal cells (MSC) or growth factors (GF). In the literature there are many studies available about their application in monotherapy, but unfortunately the healing rate doesn't exceed 90%. Polytherapy seems to be a logical option to improve the healing rate, nevertheless, there are not still extensive studies that validate this strategy and moreover, some questions are not resolved.

A 3D-printed polycaprolactone/ß-tricalcium phosphate mandibular prosthesis: A pilot animal study.

OBJECTIVE: In this study, we assessed the effectiveness of a tonsil-derived mesenchymal stem cell (TMSC)-transplanted polycaprolactone/beta-tricalcium phosphate prosthesis (specifically designed for easier fixing and grafting with a single scaffold) on rabbit mandible osteogenesis. METHODS: The mandibles of 18 rabbits were exposed, and 10 × 8-mm bone defects were made. Two rabbits did not receive implants; four were reconstructed with the scaffold control (SC) (SC group); four were reconstructed with scaffolds soaked in peripheral blood (PB) (PB group); four were reconstructed with TMSC-transplanted scaffolds (TMSC group); and four were reconstructed with differentiated osteocyte-transplanted scaffolds (DOC) (DOC group). Each rabbit was sacrificed 12 weeks after surgery, and the area of new bone formation was investigated by mechanical testing, histology, and micro-computed tomography. RESULTS: More extended and denser new bone masses were observed in the TMSC and DOC groups, although fibrosis and vascular formation levels were similar in all groups, suggesting that the dual-structured scaffold alone provides a good environment for bone attachment and regeneration. The bone volumes of representative scaffolds from the SC, PB, TMSC, and DOC groups were 43.12, 48.35, 53.10, and 57.44% of the total volumes, respectively. CONCLUSION: The design of the scaffold resulted in effective osteogenesis, and TMSCs showed osteogenic potency, indicating that their combination could enable effective bone regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 130:358-366, 2020.

Are culture-expanded autogenous bone cells a clinically reliable option for sinus grafting?

OBJECTIVES: This prospective clinical study was designed to examine the healing process during the first 12 months after sinus grafting (SG) with autogenous culture-expanded bone cells (ABC) and bovine bone mineral (BBM) histomorphometrically and radiologically. MATERIAL AND METHODS: Twenty-two sinuses of 12 patients (mean age 56.2+/-9.3 years) were grafted. Four weeks before, SG bone biopsies were obtained with a trephine burr and the bone cells were isolated and expanded. Every sinus was grafted with BBM and ABC. After 6 months, a biopsy was taken from each sinus and implants (n=82) were placed. These were uncovered after another 6 months and fitted with dentures. The percent newly formed bone (NB) and the NB-to-BBM contact area were determined on undecalcified histologic sections. The sinus graft volume was evaluated by dental CT after SG (CT 1), after implant placement (CT 2) and after implant uncovery (CT 3). RESULTS: Postoperative healing was uneventful. The NB was 17.9+/-4.6% and the contact area 26.8+/-13.1%. The graft volume (in mm(3)) was 2218.4+/-660.9 at the time of CT 1, 1694+/-470.4 at the time of CT 2 and 1347.9+/-376.3 at the time of CT 3 (P<.01). Three implants were lost after uncovery. Reimplantation and prosthodontic rehabilitation were successful throughout. CONCLUSIONS: These results suggest that SG with ABC and BBM in a clinical setting provides a bony implant site which permits implant placement and will tolerate functional loading.

Guided bone regeneration with the combined use of resorbable membranes and autogenous drilling dust or xenografts for the treatment of dehiscence-type defects around implants: an experimental study in dogs.

PURPOSE: This study aimed to measure the numbers of viable bone cells present in autogenous drilling dust (ADD) and mandibular particulated bone (MPB) and to histomorphometrically compare the effects of the combined use of resorbable membranes and ADD or xenografts for the treatment of dehiscence-type defects around implants. MATERIALS AND METHODS: The left mandibular premolars were extracted from 4 adult beagle dogs. After a 3-month healing period, 4 standardized bone defects were prepared on each mandible, and 1 implant was placed per defect. The 4 sites in each dog were allocated to 4 different treatment groups: 1 site received ADD alone (ADD); 1 site received a Cytoplast membrane supported by ADD (ADD+CP); 1 site received BioCera alone (BC); and the final site received a Cytoplast membrane supported by BioCera (BC+CP). Each animal received a series of 3 bone labels. Three months following these regenerative surgeries, animals were sacrificed and histomorphometric examinations were carried out. In addition, in 3 of the 4 dogs at the time of regenerative surgery, ADD was obtained using implant drills (group 1), MPB was obtained using a fissure bur and rongeur (group 2), and 1.0 cm3 of each was then cultured. Cultured cell counts and osteocalcin synthesis analysis using reverse transcription-polymerase chain reaction were performed on cells from these 2 groups at 4 and 9 weeks after regenerative surgery. Alkaline phosphatase activity (ALP) was measured at 9 weeks in both groups. RESULTS: MPB revealed greater cell counts than ADD after 4 and 9 weeks. Cells stained positively for ALP and osteocalcin in both groups. Fluorescence microscopy showed 22.4% bone formation with ADD+CP, 17.8% with BC+CP, 13.1% with ADD, and 6.4% with BC at 8 weeks. Bone regeneration heights were 2.0 mm with AD, 1.9 mm with ADD+CP, 1.7 mm with BC+CP, and 1.3 mm with BC. Bone regeneration areas measured 1.0 mm2 with ADD, 0.9 mm2 with ADD+CP, 0.6 mm2 with BC+CP, and 0.3 mm2 with BC. Bone-to-implant contacts were 53.1% with ADD, 46.6% with ADD+CP, 44.1% with BC, and 33.7% with BC+CP. CONCLUSIONS: ADD appears to be a useful material for closing dehiscence-type defects, and the use of a membrane was not found to affect bone formation during the treatment of dehiscence-type defects around implants in this study. However, larger studies are needed before fully endorsing its widespread use.

Repair of porcine articular cartilage defect with a biphasic osteochondral composite.

Autologous chondrocyte implantation (ACI) has been recently used to treat cartilage defects. Partly because of the success of mosaicplasty, a procedure that involves the implantation of native osteochondral plugs, it is of potential significance to consider the application of ACI in the form of biphasic osteochondral composites. To test the clinical applicability of such composite construct, we repaired osteochondral defect with ACI at low cell-seeding density on a biphasic scaffold, and combined graft harvest and implantation in a single surgery. We fabricated a biphasic cylindrical porous plug of DL-poly-lactide-co-glycolide, with its lower body impregnated with beta-tricalcium phosphate as the osseous phase. Osteochondral defects were surgically created at the weight-bearing surface of femoral condyles of Lee-Sung mini-pigs. Autologous chondrocytes isolated from the cartilage were seeded into the upper, chondral phase of the plug, which was inserted by press-fitting to fill the defect. Defects treated with cell-free plugs served as control. Outcome of repair was examined 6 months after surgery. In the osseous phase, the biomaterial retained in the center and cancellous bone formed in the periphery, integrating well with native subchondral bone with extensive remodeling, as depicted on X-ray roentgenography by higher radiolucency. In the chondral phase, collagen type II immunohistochemistry and Safranin O histological staining showed hyaline cartilage regeneration in the experimental group, whereas only fibrous tissue formed in the control group. On the International Cartilage Repair Society Scale, the experimental group had higher mean scores in surface, matrix, cell distribution, and cell viability than control, but was comparable with the control group in subchondral bone and mineralization. Tensile stress-relaxation behavior determined by uni-axial indentation test revealed similar creep property between the surface of the experimental specimen and native cartilage, but not the control specimen. Implanted autologous chondrocytes could survive and could yield hyaline-like cartilage in vivo in the biphasic biomaterial construct. Pre-seeding of osteogenic cells did not appear to be necessary to regenerate subchondral bone.

Cells seeded on MBG scaffold survive impaction grafting technique: Potential application of cell-seeded biomaterials for revision arthroplasty.

The objective of this study was to investigate the potential application of cell-seeded biomaterials for revision arthroplasty and the reconstruction of major joints using the impaction grafting technique. Using morselized cancellous bone graft as a porous scaffold, MG63 cells were seeded on the scaffold and impacted into an acetabulum cup model using a mechanical device constructed from data obtained during impaction grafting by an orthopedic surgeon. Immediately after impaction, cells were trypsinized from the scaffold and processed for cell survival rates using the double-stranded DNA PicoGreen assay. Significant reductions in viable cells were observed between the fifth impact and both the first and second impacts (p < 0.01 and p < 0.05, respectively). Cell survival rate was 21.5% after five impacts. The biological performance of cell-seeded biomaterials may be enhanced by these surviving cells. Compared to allograft bone that is not osteogenic, a cell-seeded biomaterial might also be a suitable substitute for allograft bone for major joint reconstruction at revision arthroplasty.

Total knee replacement in previous recipients of fresh osteochondral allograft transplants.

BACKGROUND: Fresh osteochondral allograft transplantation is a treatment option for young patients with osteochondral lesions of the knee. The present study evaluated the surgical complexity of, and the prevalence of complications related to, total knee arthroplasty in patients who had had a previous osteochondral graft transplantation. METHODS: A retrospective analysis was performed on thirty-three consecutive patients (thirty-five knees) who underwent total knee arthroplasty from 1974 to 2000 after having had a previous transplantation of a fresh osteochondral allograft into the same knee. The mean duration of follow-up was ninety-two months. Perioperative data were analyzed with regard to etiology, preoperative impairment, intraoperative technical complications, early and late postoperative complications, and postoperative functional and subjective outcomes. The Knee Society clinical rating system was used for clinical evaluation beginning in 1990. RESULTS: Four knees required additional techniques for exposure. Three knees required stemmed components, one knee required a tibial augment, and two knees required morselized grafts. The mean Knee Society objective score (available for eighteen knees) improved from 34.7 preoperatively to 87.9 at the time of the latest follow-up, and the mean Knee Society function score improved from 45 to 82. The mean range of motion of all knees improved from 85 degrees to 105 degrees . Six of the thirty-five knees underwent revision total knee arthroplasty because of aseptic loosening, with two knees being revised within two years after the index total knee arthroplasty. CONCLUSIONS: Total knee arthroplasty after previous fresh osteochondral allograft transplantation provides improvements in knee function and range of motion, with manageable technical difficulties. Compared with routine total knee arthroplasty, an increased rate of early revision can be expected.

HIF-1α Promotes Glutamine-Mediated Redox Homeostasis and Glycogen-Dependent Bioenergetics to Support Postimplantation Bone Cell Survival.

Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general.

[The fate of donor bone cells in different diameter bone grafts: an experiment study].

OBJECTIVE: To investigate the fate of donor bone cells in bone grafts of different diameters during repairing bone defects. METHODS: One hundred sixteen female syngeneic inbred DA rats were established as radial defect model of 3 kinds: structural bone grafting group (n = 56), morselized bone grafting group (n = 56), and blank group (without bone grafting, n = 4), to be used as receptors The ilia of 58 male inbred DA rats, as donors, were harvested and made into structural bone grafts 2 mm in diameter and morselized bone grafts 0.3 - 0.5 mm in diameter to be transplanted into the radial defects of the female receptors. One and four days, and 1, 2, 4, 6, and 8 weeks after transplantation DNA was extracted from the grafted bones and polymerase chain reaction (PCR) specific for the sex-determining region of Y-chromosome (Sry) was performed to observe the presence and relative amount of Y-chromosome originating from the bone grafts, expression of the sex-determining gene Sry in the receptors' bones and the histology of the receptors' bones. RESULTS: In structural bone grafting group, the amounts of Sry-specific bands decreased in the early time and disappeared 1 week after transplantation, and re-appeared 4 weeks after transplantation with the amount increasing with the lapse of time. In morselized bone grafting group, Sry-specific bands were detected all the time but their amounts decreased with the lapse of time. At each time point, morselized bone graft provided more living osteocytes with better effect of osteogenesis in comparison with the structural bone graft. CONCLUSION: Bone grafts of different diameters provide donor cells in repairing bone defects. Having more surviving osteocytes, morselized bone grafts may accelerate the healing pf bone defects, thus providing a new and effective method to repair bone defects and spinal fusion clinically.

Regulation of Bone Homeostasis by Osteocytes.

New data concerning the function of osteocytes as the central regulators of bone homeostasis are briefly outlined. It is established that osteocytes are the main target cells for parathormone. They are a rich source of sclerostin, the main inhibitor of osteoblast activity, and of the RANKL cytokine, the most important regulator of osteoclastogenesis. Under shear stress causing microinjury, osteocytes enter programmed cell death (apoptosis) and osteocyte apoptosis is a signal for nearby healthy osteocytes to activate osteoclasts to resorb bone.

Studies of the behaviour of allogeneic cancellous bone grafts in inbred rats.

The degree of new bone formation in isografts and allografts has been assessed by a radiotracer technique. Allografts used have been either H-1-identical or H-1-disparate with the recipient. It was found that new bone formation is curtailed within the first 3 weeks after grafting. This curtailment was significantly greater in those donor-recipient combinations which were H-1-identical but disparate for many weak locus antigens than those in which H-1 disparity existed with only a few minor locus differences. There was no late phase (i.e., host-derived) osteogenesis in the combination with H-1 identity, but multiple minor antigen disparities. In isografts and H-1-disparate grafts significant late phase osteogenesis was found in about 30% of recipients. Resorption of the graft was found to be significantly impaired in both H-1-identical and disparate grafts in comparison with isografts. There was a significant negative correlation between weight of the graft and degree of osteogenesis in isografts during the first 2 weeks after grafting, and also during the later phase (6-8 weeks). A similar negative correlation was found during the late phase in H-1-disparate grafts with few minor antigen disparities, but no correlation was found at any time in H-1-identical grafts with multiple minor antigen disparities.

Engineered osteochondral grafts using biphasic composite solid free-form fabricated scaffolds.

Tissue engineering has provided an alternative to traditional strategies to repair cartilage damaged by injury or degenerative disease. A successful strategy to engineer osteochondral tissue will mimic the natural contour of the articulating surface, achieve native mechanical properties and functional load-bearing ability, and lead to integration with host cartilage and underlying subchondral bone. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match articular geometry. The objective of this study was to utilize materials and biological factors in an integrated approach to regenerate a multitissue interface. Biphasic composite scaffolds manufactured by IBD and SFF fabrication were used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-L-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein 7 (BMP-7) in the ceramic phase and fully differentiated chondrocytes in the polymeric phase. After subcutaneous implantation into mice, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects.

The use of in situ bone marrow stem cells for the treatment of various degenerative diseases.

The potential for tissue repair and regeneration is encouraging in the light of novel research on the plasticity of adult stem cells. Intense research efforts over the last 3 years have provided solid evidence for the continuous generation of many types of tissue cells from adult stem cells as a normal part of our physiology throughout development and adult life in mammals, including humans. This opens new therapeutic avenues for many clinical problems and provides alternative opportunities at a time when much attention has been brought to the issue of using embryonic stem cells for research purposes and for the development of treatments for various diseases. Embryonic stem cells are pluripotent cells characterized by nearly unlimited self-renewal and differentiation capacity. However, evidence has accumulated over the past few years, indicating that adult bone marrow stem cells might have pluripotent properties similar to those of embryonic stem cells. Based on a review of the literature we propose the hypothesis that in situ mobilization of stem cells from the bone marrow and their migration to various tissues is a normal physiological process of regeneration and repair and that therapeutic benefits can be generated with less invasive regimens than the removal and re-injection of stem cells, through the stimulation of normal stem cell migration. We further propose that effort should be made to identify natural compounds characterized by their ability to augment this normal process of mobilization and re-colonization of bone marrow stem cells for the potential treatment of various degenerative diseases.

Cell transplantation from limb allografts.

A murine model of skeletal tissue transplantation was developed to study the allograft rejection process in mice for limb allograft transplantation. Muscle, bone, and skin have been shown to be strong antigenic stimuli in vascularized allograft models, and cells from these sources were used for transplantation. Using enzymatic digestion, keratinocytes, myocytes, and osteocytes were harvested from B10.A mice tissues, dissociated into single cells, and then grown in culture for 14 to 21 days. Each cell type was marked with an intracellular fluorescent marker before transplantation of the cells into pockets in the rectus abdominis muscle of a syngenic host. All cell types remained viable and were detectable 2 weeks following transplantation when examined histologically and observed under a fluorescent microscope. Transplanted osteocytes were found to produce bone 8 weeks following transplantation. These results demonstrate that individual cells transplanted into muscle pockets survive and have the ability to produce extracellular matrix in this mouse model of skeletal tissue transplantation. Use of this model will allow transplantation of the cellular components comprising limb allografts to study the relative antigenicities and the rejection of the separate cells with the advanced immunologic techniques available for mice. A better understanding of immunologic responses to these individual tissue components may enable specific donor tissue or host immune modification to achieve skeletal tissue transplantation without immunosuppression. These findings are particularly valuable to the field of tissue engineering where allogeneic cells may be used in cell/polymer constructs for reconstructive procedures.

Bone formation in trabecular bone cell seeded scaffolds used for reconstruction of the rat mandible.

This study tested whether different in vitro cultivation techniques for tissue-engineered scaffolds seeded with human trabecular bone cells affect in vivo bone formation when implanted into critical-size defects in rat mandibles. Human trabecular cells were isolated and seeded into three types of scaffolds (porous CaCO(3), mineralized collagen, porous tricalcium phosphate). Four in vitro groups were produced: empty control scaffolds incubated with cell culture medium for 24 h; scaffolds seeded with trabecular bone cells, cultivated under static conditions for 24 h; scaffolds seeded with trabecular bone cells, cultivated for 14 days under static conditions; scaffolds seeded with trabecular bone cells, cultivated for 14 days in a continuous flow perfusion bioreactor. The scaffolds were implanted press fit into non-healing defects, 5 mm diameter, in rat mandibles. After 6 weeks the presence of human cells was assessed; none were detected. Histomorphometric evaluation showed that neither seeding human trabecular bone cells nor the culturing technique increased the amount of early bone formation compared with the level provided by osteoconductive bone ingrowth from the defect edges. It is concluded that human bone marrow stroma cells in tissue-engineered scaffolds and associated in vitro technology are difficult to test in the mandible in animal models.

[Value of osteo-chondral paste autologous transplantation in experimental cartilage defects reconstruction. Part IV--Microscopic analysis of cellularity and of traits of necrosis in the defect-filling tissue]. / Wartosc autologicznych przeszczepów miazgi chrzestno-kostnej w doswiadczalnym leczeniu uszkodzen chrzastki stawowej. Czesc IV--ocena mikroskopowa komórkowosci i obecnosci cech martwicy w tkance wypelniajacej ubytek.

INTRODUCTION: Restricted potential of cartilage to regeneration following trauma induced search for new techniques of articular cartilage repair. Present study aimed at defining in experimental conditions of autologous osteochondral paste value by evaluation of cellularity and presence of necrosis in the defect-filling tissue. MATERIAL AND METHODS: Full thickness defect (IV(o)--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale. RESULTS: In evaluation of cellularity in the developed repair tissue, in long-term observations best results were obtained in the group of osteochondral paste (A) and in the group of unfilled defect (C). Following 12 weeks of observation cellularity of the repair tissue in the groups amounted to around 95%, as compared to the surrounding healthy cartilage. In evaluation of necrotic traits in the newly developed tissue, their highest intensity after 4 weeks of observation was noted in the group with periosteum covered osteochondral paste (B), and lower but still significant in the group in which paste alone was applied (A). This resulted from massive dying out of the grafted bone elements, and in the case of group B, also of the periosteal cells in the covering flap and of certain number of chondrocytes composing the graft. The trait was more objectively evaluated in long-term observations, when necrotic foci involved only elements of the proper repair tissue. After 12 weeks of observation degenerative changes showed the least pronounced intensity in the group with the paste alone (A). Following 4 weeks the intensity was least pronounced in the group with the unfilled defect (C), i.a. due to the absence of graft with the decomposing bone elements. However, with elapsing time degenerative lesions intensified in the group, pointing to the low stability of the developed tissue. The results indicated that application of the osteochondral paste in treatment of cartilage defects yields a stable, highly cellular tissue, resistant to time and trauma.