High-Speed Fluorescence Imaging Corroborates Biological Data on the Influence of Different Nozzle Types on Cell Spray Viability and Formation.

Treating severe dermal disruptions often presents significant challenges. Recent advancements have explored biological cell sprays as a promising treatment, but their success hinges on efficient cell delivery and complete wound coverage. This requires a good spray distribution with a small droplet size, high particle number, and ample surface coverage. The type of nozzle used with the spray device can impact these parameters. To evaluate the influence of different nozzles on spray characteristics, we compared air-assisted and unassisted nozzles. The unassisted nozzle displayed small particle size, high particle number, good overall coverage, high cell viability, preserved cell metabolic activity, and low cytotoxicity. Air-assisted nozzles did not perform well regarding cell viability and metabolic activity. Flow visualization analysis comparing two different unassisted nozzles using high-speed imaging (100 kHz frame rate) revealed a tulip-shaped spray pattern, indicating optimal spray distribution. High-speed imaging showed differences between the unassisted nozzles. One unassisted nozzle displayed a bi-modal distribution of the droplet diameter while the other unassisted nozzle displayed a mono-modal distribution. These findings demonstrate the critical role of nozzle selection in successful cell delivery. A high-quality, certified nozzle manufactured for human application omits the need for an air-assisted nozzle and provides a simple system to use with similar or better performance characteristics than those of an air-assisted system.

In Vivo Engineering and Transplantation of Axially Vascularized and Epithelialized Flaps in Rats.

The arteriovenous loop (AVL) model allows the in vivo engineering of axially vascularized flaps, the so-called AVL flaps. Although AVL flaps can be transplanted microsurgically to cover tissue defects, they lack an epithelial layer on the surface. Therefore, the objective of this study was to engineer axially vascularized AVL flaps with an accompanying epithelial layer for local defect reconstruction. In this study, AVLs were established in 20 male Lewis rats. Minimally invasive injection of keratinocytes onto the surface of the AVL flaps was performed on postoperative day (POD) 21. AVL flaps were explanted from 12 rats on POD 24 or POD 30, then the epithelium formed by the keratinocytes on the surface of the flaps was evaluated using immunofluorescence staining. In six other rats, the AVL flap was locally transposed to cover a critical defect in the rats' leg on POD 30 and explanted for analysis on POD 40. In two control rats, sodium chloride was applied instead of keratinocytes. These control flaps were also transplanted on POD 30 and explanted on POD 40. Our results revealed that 3 days after keratinocyte application, a loose single-layered epithelium was observed histologically on the AVL flaps surface, whereas after 9 days, a multilayered and structured epithelium had grown. The epithelium on the transplanted AVL flaps showed its physiological differentiation when being exposed to an air-liquid interface. Histologically, a layered epithelium identical to the rats' regular skin was formed. In the sodium chloride control group, no epithelium had been grown. This study clearly demonstrates that axially vascularized AVL flaps can be processed in the subcutaneous chamber by minimally invasive injection of keratinocytes. Thus, AVL flaps with an intact epithelial layer were engineered and could be successfully transplanted for local defect coverage in a small animal model.

Peracetic acid sterilized tendon and ligament allografts for knee reconstruction : For anterior cruciate ligament (ACL), posterior cruciate ligament (PCL) and complex knee surgery.

BACKGROUND: The use of allografts and autografts has been met with mixed views on whether allografts are a suitable alternative to autografts. QUESTION: We aimed to investigate if chemically sterilized allografts show similar rerupture rates to those reported in the literature for allografts and autografts in anterior (ACL) and posterior cruciate ligaments (PCL) and complex knee surgery. MATERIALS AND METHODS: Retrospective data on knee reconstructions performed between 2011 and 2015 with tendon/ligamnet allografts sterilized with peracetic acid were collected in the form of a questionnaire. The inclusion criteria of 2 years for each patient were met by 38 patients, representing 22 ACL reconstructions, 5 PCL reconstructions, 3 OTHER surgeries, including the Larson technique and medial patellofemoral ligament (MPFL) reconstruction and 8 COMPLEX surgeries. The main endpoints were rerupture and complication rate. Secondary endpoints included stability of the knee (Lachman test, Pivot shift test) and the range of motion. RESULTS: The rerupture rate was 7.9% (3 grafts). Reruptures only occurred in the ACL group. No reruptures were observed in the PCL, OTHER and COMPLEX surgery groups. Stability improved significantly after surgery and the range of motion returned to values similar to that of healthy knees. CONCLUSIONS: Tendon allografts sterilized with peracetic acid show promising low rerupture rates and good clinical scores and the results are comparable to the literature on autografts and other allografts.

Anatomical acromioclavicular joint stabilization with chemically sterilized tendon allografts: A retrospective study.

Background: The purpose of this retrospective study was to analyze whether chemically sterilized tendon allografts perform as well as other non-sterilized allografts and autografts as described in the literature for anatomical acromioclavicular joint stabilization for the treatment of Rockwood III-V. Allografts are still described as a factor for higher re-rupture rates. Methods: Retrospective data were collected from 21 acromioclavicular joint stabilizations performed by a single surgeon and performed between 2011 and 2014 using sterilized semitendinosus allografts. The primary endpoints were re-rupture and complication rates. Secondary endpoints were AC-joint stability, pain level, return to work and sport and the range of motion. Results: No re-ruptures occurred during the mean follow-up time of 33 months. Zero complications occurred directly after surgery, but three complications later than three weeks after surgery. All cases resolved without further surgery. After surgery, stability significantly improved for all patients. Post-surgery, 19 patients had stable acromioclavicular joints and only two patients showed minor instabilities. Range of motion returned to the range of the healthy shoulders for all patients. Conclusion: Chemically sterilized semitendinosus allograft use for anatomic AC-joint stabilization is equivalent to the use of other allografts or autografts and required no hardware removal. No donor age or graft size dependence was observed, due to zero re-ruptures.

Analysis of the Ability of Different Allografts to Act as Carrier Grafts for Local Drug Delivery.

Bone defects and infections pose significant challenges for treatment, requiring a comprehensive approach for prevention and treatment. Thus, this study sought to evaluate the efficacy of various bone allografts in the absorption and release of antibiotics. A specially designed high-absorbency, high-surface-area carrier graft composed of human demineralized cortical fibers and granulated cancellous bone (fibrous graft) was compared to different human bone allograft types. The groups tested here were three fibrous grafts with rehydration rates of 2.7, 4, and 8 mL/g (F(2.7), F(4), and F(8)); demineralized bone matrix (DBM); cortical granules; mineralized cancellous bone; and demineralized cancellous bone. The absorption capacity of the bone grafts was assessed after rehydration, the duration of absorption varied from 5 to 30 min, and the elution kinetics of gentamicin were determined over 21 days. Furthermore, antimicrobial activity was assessed using a zone of inhibition (ZOI) test with S. aureus. The fibrous grafts exhibited the greatest tissue matrix absorption capacity, while the mineralized cancellous bone revealed the lowest matrix-bound absorption capacity. For F(2.7) and F(4), a greater elution of gentamicin was observed from 4 h and continuously over the first 3 days when compared to the other grafts. Release kinetics were only marginally affected by the varied incubation times. The enhanced absorption capacity of the fibrous grafts resulted in a prolonged antibiotic release and activity. Therefore, fibrous grafts can serve as suitable carrier grafts, as they are able to retain fluids such as antibiotics at their intended destinations, are easy to handle, and allow for a prolonged antibiotic release. Application of these fibrous grafts can enable surgeons to provide longer courses of antibiotic administration for septic orthopedic indications, thus minimizing infections.

One Stage Masquelets Technique: Evaluation of Different Forms of Membrane Filling with and without Bone Marrow Mononuclear Cells (BMC) in Large Femoral Bone Defects in Rats.

The classic two-stage masquelet technique is an effective procedure for the treatment of large bone defects. Our group recently showed that one surgery could be saved by using a decellularized dermis membrane (DCD, Epiflex, DIZG). In addition, studies with bone substitute materials for defect filling show that it also appears possible to dispense with the removal of syngeneic cancellous bone (SCB), which is fraught with complications. The focus of this work was to clarify whether the SCB can be replaced by the granular demineralized bone matrix (g-DBM) or fibrous demineralized bone matrix (f-DBM) demineralized bone matrix and whether the colonization of the DCD and/or the DBM defect filling with bone marrow mononuclear cells (BMC) can lead to improved bone healing. In 100 Sprague Dawley rats, a critical femoral bone defect 5 mm in length was stabilized with a plate and then encased in DCD. Subsequently, the defect was filled with SCB (control), g-DBM, or f-DBM, with or without BMC. After 8 weeks, the femurs were harvested and subjected to histological, radiological, and biomechanical analysis. The analyses showed the incipient bony bridging of the defect zone in both groups for g-DBM and f-DBM. Stability and bone formation were not affected compared to the control group. The addition of BMCs showed no further improvement in bone healing. In conclusion, DBM offers a new perspective on defect filling; however, the addition of BMC did not lead to better results.

The Induced Membrane Technique-The Filling Matters: Evaluation of Different Forms of Membrane Filling with and without Bone Marrow Mononuclear Cells (BMC) in Large Femoral Bone Defects in Rats.

The Masquelet technique is used to treat large bone defects; it is a two-stage procedure based on an induced membrane. To improve the induced membrane process, demineralized bone matrix in granular (GDBM) and fibrous form (f-DBM) was tested with and without bone marrow mononuclear cells (BMC) as filling of the membrane against the gold standard filling with syngeneic cancellous bone (SCB). A total of 65 male Sprague-Dawley rats obtained a 5 mm femoral defect. These defects were treated with the induced membrane technique and filled with SCB, GDBM, or f-DBM, with or without BMC. After a healing period of eight weeks, the femurs were harvested and submitted for histological, radiological, and biomechanical analyses. The fracture load in the defect zone was lower compared to SCB in all groups. However, histological analysis showed comparable new bone formation, bone mineral density, and cartilage proportions and vascularization. The results suggest that f-DBM in combination with BMC and the induced membrane technique cannot reproduce the very good results of this material in large, non-membrane coated bone defects, nevertheless it supports the maturation of new bone tissue locally. It can be concluded that BMC should be applied in lower doses and inflammatory cells should be removed from the cell preparation before implantation.

Fibrous Demineralized Bone Matrix (DBM) Improves Bone Marrow Mononuclear Cell (BMC)-Supported Bone Healing in Large Femoral Bone Defects in Rats.

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect.

Influence of peracetic acid-ethanol sterilisation on the biomechanical properties of human meniscus transplants.

PURPOSE: Meniscus allograft transplantation (MAT) is a possible treatment for patients suffering with pain after meniscectomy. Here, peracetic acid (PAA) sterilised meniscus transplants were investigated on whether they would provide an adequate alternative to fresh-frozen transplants in their viscoelastic and mechanical properties. METHODS: In this analysis, 31 menisci donors (26 male and 5 female) were included. The average donor age was 49.87 years, ranging from 32 to 65 years. Menisci of matched pairs of knees underwent chemical sterilisation while counterparts were left fresh-frozen. Stiffness and load to failure were determined via suture retention. Further menisci were analysed while attached to the tibial bone block using a novel test device to mimic physiological load distribution. Meniscus relaxation, stiffness and failure loads were determined. Histology and biphasic properties of the menisci were examined and results were analysed using paired t-tests. RESULTS: A novel custom built test device allowed the application of physiological loads for suture retention testing and revealed no significant differences between PAA sterilised (14.85 ± 4.46 N/mm, 50.49 ± 17.01 N) and fresh-frozen (18.26 ± 4.46 N/mm, 59.49 ± 21.07 N) regarding stiffness and failure load, respectively. Furthermore, initial 200 N loading showed significantly higher strain in sterilised menisci (18.87 ± 1.56) compared to fresh frozen (13.81 ± 1.04). Load relaxation experiments demonstrated significantly lower relaxation for sterilised menisci (77.71 ± 1.62) compared to fresh-frozen (89.11 ± 1.00, p-value < 0.0001). CONCLUSION: Peracetic acid sterilised human menisci performed equally to fresh-frozen counterparts in a suture retention test and in physiological failure testing providing an adequate alternative. However, meniscus relaxation, biphasic properties and strain were shown to be significantly different between the groups. A common problem of MAT is graft extrusion or shrinkage, therefore the parameters measured here should be considered and may influence meniscus extrusion after transplantation. LEVEL OF EVIDENCE: n/a (experimental study).

First Human Leucocyte Antigen (HLA) Response and Safety Evaluation of Fibrous Demineralized Bone Matrix in a Critical Size Femoral Defect Model of the Sprague-Dawley Rat.

Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.

Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model.

Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100 mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies. KEY MESSAGES: Bone tissue engineering. Mesenchymal stem cells derived from human adipose tissue (hASCs). Optimal cell/volume ratio of cell-seeded scaffolds. Increased osteogenesis and angiogenesis in vivo.

Comparison of three different types of scaffolds preseeded with human bone marrow mononuclear cells on the bone healing in a femoral critical size defect model of the athymic rat.

Large bone defects often pose major difficulties in orthopaedic surgery. The application of long-term cultured stem cells combined with a scaffold lead to a significant improvement of bone healing in recent experiments but is strongly restricted by European Union law. Bone marrow mononuclear cells (BMC), however, can be isolated and transplanted within a few hours and have been proven effective in experimental models of bone healing. The effectivity of the BMC-supported therapy might be influenced by the type of scaffold. Hence, we compared three different scaffolds serving as a carrier for BMC in a rat femoral critical size defect with regard to the osteogenic activity in the defect zone. Human demineralized bone matrix (DBM), bovine cancellous bone hydroxyapatite ceramic (BS), or ß-tricalcium phosphate (ß-TCP) were seeded with human BMC and hereafter implanted into critically sized bone defects of male athymic nude rats. Autologous bone served as a control. Gene activity was measured after 1 week, and bone formation was analysed histologically and radiologically after 8 weeks. Generally, regenerative gene expression (BMP2, RUNX2, VEGF, SDF-1, and RANKL) as well as bony bridging and callus formation was observed to be most pronounced in defects filled with autologous bone, followed in descending order by DBM, ß-TCP, and BS. Although DBM was superior in most aspects of bone regeneration analysed in comparison to ß-TCP and BS, the level of autologous bone could not be attained.

Anti-endotoxic and antibacterial effects of a dermal substitute coated with host defense peptides.

Biomaterials used during surgery and wound treatment are of increasing importance in modern medical care. In the present study we set out to evaluate the addition of thrombin-derived host defense peptides to human acellular dermis (hAD, i.e. epiflex(®)). Antimicrobial activity of the functionalized hAD was demonstrated using radial diffusion and viable count assays against Gram-negative Escherichia coli, Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Electron microscopy analyses showed that peptide-mediated bacterial killing led to reduced hAD degradation. Furthermore, peptide-functionalized hAD displayed endotoxin-binding activity in vitro, as evidenced by inhibition of NF-κB activation in human monocytic cells (THP-1 cells) and a reduction of pro-inflammatory cytokine production in whole blood in response to lipopolysaccharide stimulation. The dermal substitute retained its anti-endotoxic activity after washing, compatible with results showing that the hAD bound a significant amount of peptide. Furthermore, bacteria-induced contact activation was inhibited by peptide addition to the hAD. E. coli infected hAD, alone, or after treatment with the antiseptic substance polyhexamethylenebiguanide (PHMB), yielded NF-κB activation in THP-1 cells. The activation was abrogated by peptide addition. Thus, thrombin-derived HDPs should be of interest in the further development of new biomaterials with combined antimicrobial and anti-endotoxic functions for use in surgery and wound treatment.

Characterization of bone marrow mononuclear cells on biomaterials for bone tissue engineering in vitro.

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (ß-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on ß-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and ß-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo.

Primary mesenchymal stem cells in human transplanted lungs are CD90/CD105 perivascularly located tissue-resident cells.

BACKGROUND: Mesenchymal stem cells (MSC) have not only been implicated in the development of lung diseases, but they have also been proposed as a future cell-based therapy for lung diseases. However, the cellular identity of the primary MSC in human lung tissues has not yet been reported. This study therefore aimed to identify and characterise the 'bona fide' MSC in human lungs and to investigate if the MSC numbers correlate with the development of bronchiolitis obliterans syndrome in lung-transplanted patients. METHODS: Primary lung MSC were directly isolated or culture-derived from central and peripheral transbronchial biopsies of lung-transplanted patients and evaluated using a comprehensive panel of in vitro and in vivo assays. RESULTS: Primary MSC were enriched in the CD90/CD105 mononuclear cell fraction with mesenchymal progenitor frequencies of up to four colony-forming units, fibroblast/100 cells. In situ staining of lung tissues revealed that CD90/CD105 MSCs were located perivascularly. MSC were tissue-resident and exclusively donor lung-derived even in biopsies obtained from patients as long as 16 years after transplantation. Culture-derived mesenchymal stromal cells showed typical in vitro MSC properties; however, xenotransplantation into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice showed that lung MSC readily differentiated into adipocytes and stromal tissues, but lacked significant in vivo bone formation. CONCLUSIONS: These data clearly demonstrate that primary MSC in human lung tissues are not only tissue resident but also tissue-specific. The identification and phenotypic characterisation of primary lung MSC is an important first step in identifying the role of MSC in normal lung physiology and pulmonary diseases.

Mesenchymal stromal cells from primary osteosarcoma are non-malignant and strikingly similar to their bone marrow counterparts.

Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from a number of human tissues. In cancer, MSC have been implicated with tumor growth, invasion, metastasis, drug resistance and were even suggested as possible tumor-initiating cells in osteosarcoma (OS). However, MSC from OS and their possible tumor origin have not yet been thoroughly investigated. Therefore, primary OS mesenchymal progenitors and OS-derived MSC were studied. OS samples contained very high frequencies of mesenchymal progenitor cells as measured by the colony-forming unit fibroblast (CFU-F) assay (median: 1,117 colonies per 10(5) cells, range: 133-3,000, n = 6). This is considerably higher compared to other human tissues such as normal bone marrow (BM) (1.3 ± 0.2 colonies per 10(5) cells, n = 8). OS-derived MSC (OS-MSC) showed normal MSC morphology and expressed the typical MSC surface marker profile (CD105/CD73/CD90/CD44/HLA-classI/CD166 positive, CD45/CD34/CD14/CD19/HLA-DR/CD31 negative). Furthermore, all OS-MSC samples could be differentiated into the osteogenic lineage, and all but one sample into adipocytes and chondrocytes. Genetic analysis of OS-MSC as well as OS-derived spheres showed no tumor-related chromosomal aberrations. OS-MSC expression of markers related to tumor-associated fibroblasts (fibroblast surface protein, alpha-smooth muscle actin, vimentin) was comparable to BM-MSC and OS-MSC growth was considerably affected by tyrosine kinase inhibitors. Taken together, our results demonstrate that normal, non-malignant mesenchymal stroma cells are isolated from OS when MSC culture techniques are applied. OS-MSC represent a major constituent of the tumor microenvironment, and they share many properties with BM-derived MSC.

Characterization of bone marrow-derived mesenchymal stromal cells (MSC) based on gene expression profiling of functionally defined MSC subsets.

BACKGROUND AIMS: Human mesenchymal stromal cells (MSC) are promising candidates for cell therapy because of their intriguing properties (high proliferation and differentiation capacity, microenvironmental function and immune modulation). However, MSC are heterogeneous and a better understanding of the heterogeneity of the cells that form the MSC cultures is critical. METHODS: Human MSC were generated in standard cultures and stained with carboxyfluorescein succinimidyl ester (CFSE) for cell division tracking. Gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics) was utilized to characterize potential MSC subpopulations (progenitor content and differentiation capacity) and identify potential MSC subpopulation markers. RESULTS: The majority of MSC had undergone more than two cell divisions (79.7+/-2.0%) after 10 days of culture, whereas 3.5+/-0.9% of MSC had not divided. MSC were then sorted into rapidly dividing cells (RDC) and slowly/non-dividing cells (SDC/NDC). Colony-forming unit-fibroblast (CFU-F) frequencies were lowest in NDC and highest in RDC with low forward-/side-scatter properties (RDC(lolo)). Comparative microarray analysis of NDC versus RDC identified 102 differentially expressed genes. Two of these genes (FMOD and VCAM1) corresponded to cell-surface molecules that enabled the prospective identification of a VCAM1(+)/FMOD(+) MSC subpopulation, which increased with passage and showed very low progenitor activity and limited differentiation potential. CONCLUSIONS: These data clearly demonstrate functional differences within MSC cultures. Furthermore, this study shows that cell sorting based on proliferation characteristics and gene expression profiling can be utilized to identify surface markers for the characterization of MSC subpopulations.