Malleable, cryopreserved osteogenic compositions with viable cells

Viscous cryoprotectant compositions were created for subsequent combination with tissue components. A 10% (v/v) dimethyl sulfoxide (DMSO) solution was created in an isotonic, pH neutral solution with acetate and gluconate buffers. Pre-weighed quantities of sodium alginate were dissolved in the 10% DMSO solution to achieve concentrations of 1%-4% (w/v) alginate. Alginates had been pre-selected with a Brookfield viscosity specification in the range of 100-10,000 cps when tested at 2% in water at 25 degrees C.

Relative apparent viscosities were determined for each of the final cryoprotectant solutions and ranked such that 7>6>5>4>3>2>1, as shown in Table 1.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 125-1000 μm, and demineralized to <8% residual calcium content to create hydrated demineralized bone matrix (DBM). Tissue components were mixed in cancellous:DBM volume ratios of 10:3-2:1. Tissue mixtures were combined with cryoprotectants essentially identical to those of Example 1 at a cancellous:cryoprotectant volume ratio of 5:1. Tissue and cryoprotectant components were mixed to form malleable compositions with variously satisfactory cohesiveness and formability, as shown in Table 2.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 100-710 μm, demineralized to <8% residual calcium content, and lyophilized to create lyophilized DBM. Tissue components were mixed at a cancellous:DBM volume ratio of 2:1. The tissue mixture was combined with cryoprotectants essentially identical to those of Example 1 at cancellous:cryoprotectant volume ratios of 10:3-5:2. Tissue and cryoprotectant components were mixed and evaluated for cohesiveness and formability; the results are summarized in Table 3.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 100-710 μm, demineralized to <8% residual calcium content, and lyophilized to create lyophilized DBM. Lyophilized DBM was subsequently rehydrated in an isotonic, neutral pH solution and mixed with cancellous bone at a cancellous:DBM volume ratio of 10:7. The tissue mixture was combined with a cryoprotectant essentially identical to Solution G in Example 1 at a cancellous:cryoprotectant volume ratio of 10:3. Tissue and cryoprotectant components were mixed and evaluated for cohesiveness and formability; the results are summarized in Table 4.

Viscous cryoprotectant compositions were created for subsequent combination with tissue components. Pre-weighed quantities of sodium alginate having a Brookfield viscosity specification of >2000 cps when tested at 2% in water at 25 degrees C. were suspended in measured volumes of DMSO. Measured quantities of an isotonic, pH neutral solution with acetate and gluconate buffers were mixed with the alginate/DMSO suspensions to create substantially homogeneous cryoprotectant solutions with final DMSO concentrations of 5%-10% (v/v) and alginate concentrations of 2%-4% (w/v).

Relative apparent viscosities were determined for each of the final cryoprotectant solutions and ranked such that 7>6>5>4>3>2>1, as shown in Table 5.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 125-1000 μm, and demineralized to <8% residual calcium content to create hydrated DBM. Tissue components were mixed at cancellous:DBM volume ratios of 5:1-2:1. Tissue mixtures were combined with a cryoprotectant essentially identical to Solution 0 of Example 5 with the addition of 2% (w/v) human serum albumin at cancellous:cryoprotectant volume ratios of 5:1-4:1. Tissue and cryoprotectant components were mixed and evaluated for cohesiveness and formability, the results of which are summarized in Table 6.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 125-1000 μm, and demineralized to <8% residual calcium content to create hydrated DBM. Tissue components were mixed at a cancellous:DBM volume ratio of 2:1. Cryoprotectant solutions were created consisting of DMSO at 5%-10% (v/v), human serum albumin at 0%-2% (w/v), and alginate at 4% (w/v) in an isotonic, neutral pH parenteral solution. Tissue mixtures were combined with cryoprotectants at a cancellous:cryoprotectant volume ratio of 4:1. Tissue and cryoprotectant components were mixed to create substantially homogeneous malleable compositions. Compositions were frozen to −80±5° C. to cryopreserve tissue components and viable cells.

Compositions were subsequently thawed and tested for cell viability (% viable cells) and cell concentrations (cells per cc of tissue). Compositions were rinsed immediately after thawing with phosphate buffered saline to dilute and decant the viscous cryoprotectant solutions. The remaining tissue components were treated with 3 mg/ml collagenase in phosphate buffered saline at 37° C. to release cells off bone matrix for counting. Released cells were washed and resuspended in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and then stained with Trypan blue. Live (negative staining) and dead (positive staining) cells were counted with the aid of a hemocytometer and microscope. The results are summarized in Table 7.

Viable cellular cancellous bone was ground and sieved to 425-2000 μm. Cortical bone was ground, sieved to 125-1000 μm, and demineralized to <8% residual calcium content to create hydrated DBM. Tissue components were mixed at cancellous:DBM volume ratios of 5:2 to 5:3. Cryoprotectant solutions were created consisting of DMSO at 10% (v/v), human serum albumin at 2% (w/v), and alginate at 6% (w/v) in an isotonic, neutral pH parenteral solution. Alginates in this example had molecular weights (MW) between 50,000 and 150,000 g/mol. Tissue mixtures were combined with cryoprotectants at cancellous:cryoprotectant volume ratios of 5:2 to 2:1. Tissue and cryoprotectant components were mixed to create substantially homogeneous malleable compositions. Compositions were frozen to −80±5° C. to cryopreserve tissue components and viable cells.

Compositions were subsequently thawed and tested for cell viability (% viable cells), cell concentrations (cells per cc of tissue), and osteogenic potential. Compositions were rinsed immediately after thawing with phosphate buffered saline to dilute and decant the viscous cryoprotectant solutions. The remaining tissue components were treated with 3 mg/ml collagenase in phosphate buffered saline at 37° C. to release cells off bone matrix for counting. Released cells were washed and resuspended in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and then stained with Trypan blue. Live (negative staining) and dead (positive staining) cells were counted with the aid of a hemocytometer and microscope. Cells were plated and cultured in expansion medium through one passage. Cells were then switched into osteogenic medium and subsequently stained for the presence of the bone mineralization marker alkaline phosphatase. The results are summarized in Table 8.