Cell isolation from hMSSM

For isolation of cells, samples of the hMSSM were extensively rinsed with PBS solution supplemented with antibiotics, and cut into small pieces. The tissue fragments were incubated with dispase (Sigma–Aldrich, St. Louis, MO, USA, 37 °C, 1 h) to separate the epithelial lining from the membrane. The epithelial layer was separated and discarded. The remaining tissue fragments were incubated in collagenase containing solution (Collagenase type II, Sigma–Aldrich, USA) 150 U/ml + 3 mM CaCl ₂ in Hank’s balanced salt solution (HBSS) for 2 h with constant rotation. The resulting cells were counted and 5 × 10 ⁵ cells were plated in 10 cm-diameter tissue culture dishes with α-MEM medium containing 10% foetal calf serum (FCS), 2 mM l -glutamine, Pen-Strep (both 100 U/ml), (Biological Industries, Beit Haemek, Israel). To induce osteogenic differentiation in culture the cells were passaged and P1 cultures were cultured for an additional 14 days in α-MEM medium containing 10% FCS, 2 mM l -glutamine, Pen-Strep (both 100 U/ml), 100 g/ml ascorbic acid and 10 ⁻⁸ M dexamethasone (induction medium).

Alkaline phosphatase enzyme histochemistry in fresh hMSSM

To demonstrate alkaline phosphatase (ALP) activity, tissue samples were fixed in cold acetone for 1 h, embedded in paraffin, and 6 μm thick sections were reacted with Gomori’s calcium cobalt method , using β-glycerophosphate as a substrate (Sigma–Aldrich, USA) and stained with haematoxylin-eosin (H-E) for general histology.

ALP enzyme activity in cell culture

hMSSM-derived cells grown in control and in inductive medium for 3 weeks in culture, were washed twice with PBS, fixed with 4% formaldehyde in phosphate buffer, pH 7.4, and reacted for ALP using Naphthol AS phosphate as substrate and Fast Blue BB as coupler (Sigma–Aldrich, USA). Naphthol AS phosphate was dissolved in N – N ′ dimethylformamide (30 mg in 0.5 ml) and added to a 0.1% solution of Fast Blue BB salt (Sigma–Aldrich, USA) in 0.1% boric acid/sodium tetraborate buffer, pH 9. Cultures were incubated in the ALP substrate solution for 20 min at 37 °C.

ALP enzyme activity in cell culture

Additional MSSM-derived cells were grown in control medium and in inductive medium for a maximum of 18 days in 24-well culture plates. At predetermined times (4, 8, 12, 19 days) cells were washed twice with PBS, lysed with cold lysis buffer (1 mM MgCl ₂ , 0.5% Triton X100 in alkaline buffer solution) and incubated on ice for 1 h, another equivalent portion of lysis buffer was added to the sample. Cell lysates were mixed with phosphatase substrate solution (4 mg/ml p -nitrophenol phosphate ( p -NPP) in alkaline buffer Tris–HCl, pH 9.0) for 10 min at 37 °C and then returned to ice, the reaction was stopped with ethylene diamine tetra-acetic acid (EDTA)–NaOH stop solution. The samples were transferred to a 96-well plate and absorbance was read at 404 nm using an Elisa plate reader. The results were expressed as nmol p -NP/ml/min and normalized to protein content as measured by the B radford method in corresponding wells.

Reverse transcription polymerase chain reaction

Total RNA was prepared from cells (P ₁ ; induction medium) collected from cultures as described above, using a SV Total RNA Isolation kit (Promega, Madison, WI) according to the manufacturer’s instructions. Reverse transcription was carried out employing a final concentration of 0.5 μg random primers (Promega, USA)/1 μg RNA, 500 μM dNTP mix, 10 U RNase inhibitor, 40 U M-MuLV reverse transcriptase (RT) and its accompanying buffer (25 μl final volume). The RT program consisted of heating the mRNA and random primers (final volume 15 μl) for 5 min at 70 °C followed by 5 min 0 °C. A mixture of buffer, enzymes and dNTPs were added and heated to 39 °C (1 h) followed by 90 °C (10 min).

Each polymerase chain reaction (PCR) was accomplished using 100 ng cDNA, 400 nM each of sense and antisense primers ( Table 1 ), 200 μM dNTP mix, 0.4 U/reaction of Taq polymerase and its accompanying buffer (final volume 25 μl). The PCR program consisted of 5 min 94 °C denaturisation followed by 29 cycles of 94 °C (3 min), annealing (45 s), elongation (1 min) and terminating with 5 min elongation. Both RT and PCR were carried out in PTC-100™ Thermal Control (MJ Research, Waltham, MA, USA). PCR products were resolved on 1.5% agarose gels containing ethidium bromide.

Folded hMSSM in vivo implantation into athymic nude mice

Athymic nude 8-week-old mice (Harlan, Jerusalem, Israel) were used for in vivo transplantation . All animals received care in compliance with the guidelines of the local institutional Animal Care and Use Committee following National Institutes of Health Guidelines. Fresh Schneiderian membrane samples obtained from five patients, were subjected to dispase digestion, as described above, and the resulting mucosal lamina propria were folded forming pocket-like structures (0.8 cm × 0.8 cm) where the deepest periosteal-like layer was facing the inner face of the pocket. Fibrin clots, prepared by mixing mouse fibrinogen (Sigma–Aldrich, USA) (15 μl; 3.2 mg/ml in PBS) with mouse thrombin (Sigma–Aldrich, USA) (15 μl; 25 U/ml in 2% CaCl ₂ ) to form fibrin clots, were inserted inside the pocket of the folded membrane and were transplanted subcutaneously in nude mice (five animals, two transplants in each animal). Control transplants containing only the fibrin clots were similarly transplanted ectopically (five animals). Another group of folded membranes containing no fibrin clot was transplanted similarly (five animals). A midsagittal incision was performed under anaesthesia (xylazine:ketamine 1:1) in the area of the back and the folded MSSM was subcutaneously implanted ( Fig. 1 ). After surgery, the skin was sutured carefully and dressed topically with antibiotic ointment (3% syntomycin). All mice recovered well from surgery, were housed separately in plastic cages and were followed for up to 8 weeks, Food and water were supplied ad libitum .

Preparation of hMSSM folded membrane for in vivo transplantation. Fresh tissue samples were subjected to dispase digestion and the resulting lamina propria was folded, its deepest periosteal-like layer facing the inside of the pocket and fibrin clot inserted into the folded membrane. The whole construct was transplanted subcutaneously in immunodeficient nude mice for up to 8 weeks.

Histology and histomorphometry

After 8 weeks, the transplants were dissected carefully, fixed with neutral phosphate buffered, pH 7.4 10% formalin (NBF), decalcified in 10% EDTA (5 days, room temperature), dehydrated in graded ethanols (70–100%) and embedded in paraffin (Paraplast, St. Louis, USA). Serial sections (6 μm thick) were stained with H-E stain for general histology.

H-E stained sections were used for histomorphometry measurements using Image Pro plus 6 computerized analysis system (Media Cybernetics, Silver Spring, MD, USA). The amount of bone formation was expressed as a percentage of the total imaged bone area (five animals each group, five sections from each animal). Comparisons were made using unpaired two-tailed Student’s t -test, with P < 0.05 as the significant value. For the visualisation of mineralized bone, additional samples of non-decalcified tissue samples were embedded in glycol methacrylate (GMA). The harvested transplants were immersed in 10% NBF for 3 days at 4 °C, dehydrated in 70% ethanol for 72 h (three changes) and ethanol 95% for 3 h (three changes), both at RT. The samples were immersed in infiltration solution for 2 weeks at 4 °C and then embedded in Historesin embedding kit (Leica Microsystems, Heidelberg, Germany). Sections (5 μm thick) were cut using a tungsten carbide knife and were stained with Von Kossa’s technique for identification of mineral deposition.

Tetracycline labelling

In order to distinguish the new bone formation that resulted from the folded MSSM tissue transplants, mice were injected with tetracycline (20 mg/kg) 72 h prior to termination of the experiment. Tissue transplants were fixed in NBF and processed further for embedding in GMA. The non-decalcified sections were viewed with a fluorescence microscope for demonstration of newly mineralized osteoid formed during the 72 h of exposure to the tetracycline.

Statistical analysis

Comparisons of the means of histogram analysis were made using the unpaired two-tailed Student’s t -test with significant values set at P < 0.05. The results of the experiments were expressed as mean vs. the control values ± SEM.

Histochemistry of ALP enzyme activity in the intact hMSSM

hMSSM tissue sections (6 μm thick) were subjected to Gomori’s calcium cobalt method for detection of ALP enzyme activity. Positive ALP activity was observed along the deepest layer of the detached hMSSM along the former bone interface, possibly indicating an ALP-positive periosteum-like lining ( Fig. 2 A ). A few positive ALP foci were also observed in the lamina propria ( Fig. 2 C and E arrow), demonstrating an elongated structure, possibly a blood vessel (arrow). Serial sections were stained with H-E demonstrating the general histology of the hMSSM ( Fig. 2 B), showing the epithelial lining, lamina propria, and the periosteum-like layer of the whole membrane ( Fig. 2 B). The deepest periosteum-like layer was composed of few layers of polyhedric cells along the adjacent connective tissue containing typical fibroblasts ( Fig. 2 D). The pseudostratified columnar epithelium mucous membrane is shown lining the maxillary sinus lumen ( Fig. 2 F).

ALP enzyme histochemistry in hMSSM (A, C, E) and general morphology in paraffin sections stained with H-E (B, D, F). A section of hMSSM that was stained for demonstration of ALP activity. Positive ALP enzyme activity is shown in the deepest layer of hMSSM along the periosteum-like lining. Some positive foci are seen in the lamina propria (arrow) (A, B). A higher magnification of the deepest layer of the hMSSM showing strong positive ALP reaction in this area (C). Another higher magnification of the lamina propria shows an elongated structure possibly a blood vessel located in this richly vascularized lamina propria (E). A general view of hMSSM section stained with H-E including the epithelial lining, the vascularized lamina propria and the deepest periosteal-like lining (B). A higher magnification of a section stained with H-E of the deepest layer of hMSSM composed of few layers of polyhedral cells adjacent to a typical connective tissue containing elongated fibroblasts (D). A higher magnification of the epithelial lining showing pseudostratified columnar epithelium with goblet cells and the lamina propria beneath it (F).

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