Malleable, cryopreserved osteogenic compositions with viable cells

Abstract

A bone graft composition comprising a viable, osteogenic cellular material combined with a viscous cryoprotectant that includes a penetrating cryoprotective agent and a non-penetrating cryoprotective agent. The viscosity of the cryoprotectant is such that the composition is malleable, cohesive and capable of being formed into desired shapes.

Description

FIELD

This application relates to a bone graft composition, useful in surgical applications, comprising viable cellular material combined with a viscous cryoprotectant.

SUMMARY

According to an exemplary embodiment, the bone graft composition comprises a viable, osteogenic cellular material combined with a viscous cryoprotectant that includes a penetrating cryoprotective agent and a non-penetrating cryoprotective agent. According to an exemplary embodiment, the viscosity of the cryoprotectant is such that the composition is malleable, cohesive and capable of being formed into desired shapes. According to another exemplary embodiment, the osteogenic cellular material includes viable mesenchymal stem cells. According to yet another embodiment, the osteogenic composition includes at least one of demineralized cortical bone, demineralized cancellous bone, growth factors, bone marrow, BMP-2, BMP-4, BMP-7, or a combination thereof. The characteristics of viscous cryoprotectant allow the composition to be frozen and subsequently thawed and implanted into a patient in need thereof while preserving the viability of the mesenchymal stem cells in the composition.

According to one aspect, the viable osteogenic cellular material is autogenous bone matrix having a population of endogenous osteopotent and/or osteogenic cells. According to another aspect, the viable osteogenic cellular material is allogeneic bone matrix having a population of endogenous osteopotent and/or osteogenic cells. The viable osteogenic cellular material may be substantially depleted of blood cells. The cellular material may include mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, synovium, synovial fluid, dental pulp and/or umbilical cord origin.

According to another aspect, non-penetrating cryoprotective agent is one of alginate, hyaluronic acid, hydroxyethyl starch, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, chitosan, glycerol, or a combination thereof. The penetrating cryoprotective agent is one of dimethyl sulfoxide, glycerol, propylene glycol, ethylene glycol, propanediol, or a combination thereof. According to another exemplary embodiment, the bone graft composition further comprises a scaffold material. For example, the scaffold material is one of non-demineralized, partially demineralized and demineralized cortical bone matrix; nondemineralized, partially demineralized and demineralized cancellous bone matrix; hydroxyapatite, tri-calcium phosphate, calcium sulfate, collagen or a combination thereof.

According to yet another exemplary embodiment, the viable osteogenic cellular material comprises particles cohesively bound by the viscous cryoprotectant. Alternatively, the viable osteogenic cellular material may be coated or encapsulated by the viscous cryoprotectant.

DETAILED DESCRIPTION

Aspects of the invention are disclosed in the following description. Alternate embodiments may be devised without departing from the spirit or the scope of the invention. Additionally, well-known elements of the invention will not be described in detail or omitted so as not to obscure the relevant details of the invention.

Example 1

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.

TABLE 1
Cryoprotectant	Alginate	Alginate	Relative
Solution ID	Concentration	Viscosity Spec	Viscosity
A	1%	100-300	cps	1
B	2%	100-300	cps	2
C	4%	100-300	cps	5
D	1%	>2000	cps	3
E	1.5%	>2000	cps	4
F	2%	>2000	cps	6
G	4%	>2000	cps	7

Example 2

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.

TABLE 2
Cancellous:DBM	Cryoprotectant	Cancellous:Cryo	Cohesiveness/
(v:v)	Solution ID	(v:v)	Formability
10:3	D	5:1	poor
10:3	E	5:1	poor
10:3	C	5:1	fair
10:3	F	5:1	fair
2:1	F	5:1	fair
2:1	G	5:1	good

Example 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. 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 mixes and evaluated for cohesiveness and formability; the results are summarized in Table 3.

TABLE 3
Cancellous:DBM	Cryoprotectant	Cancellous:Cryo	Cohesiveness/
(v:v)	Solution ID	(v:v)	Formability
2:1	G	10:3	fair
2:1	G	5:2	good

Example 4

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.

TABLE 4
Cancellous:DBM	Cryoprotectant	Cancellous:Cryo	Cohesiveness/
(v:v)	Solution ID	(v:v)	Formability
10:7	G	10:3	good

Example 5

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. IDC-2531

	TABLE 5
	Cryoprotectant	Alginate	DMSO	Relative
	Solution ID	Concentration	Concentration	Viscosity
	H	2%	5%	1
	I	2.5%	5%	2
	J	3%	5%	4
	K	4%	5%	6
	L	2%	10%	3
	M	2.5%	10%	5
	N	3%	10%	6
	O	4%	10%	7

Example 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 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.

TABLE 6
Cancellous:DBM	Cancellous:Cryo	Cohesiveness/
(v:v)	(v:v)	Formability
5:1	5:1	fair
4:1	5:1	fair
3:1	5:1	good
2:1	5:1	good
5:1	4:1	fair
4:1	4:1	fair
3:1	4:1	good
2:1	4:1	better

Example 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 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.

TABLE 7
		Human Serum
	DMSO	Albumin	Avg.	Avg. Cell
Composition	Concentration	Concentration	Cell	Count
ID	(v/v)	(w/v)	Viability	(cells/cc)
A	10%	0%	76.4%	4.154,500
B	7.5%	0%	74.7%	3,787,000
C	5%	0%	77.2%	4,399,500
D	10%	2%	76.8%	4,301,500
E	7.5%	2%	80.6%	4,063,500
F	5%	2%	77.1%	3,279,500

Example 8

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. IDC-28,T 1 Table 8:

TABLE 8
				Avg.
			Avg.	Cell	Alk.
Composition	Alginate	Cancellous:Cryo	Cell	Count	Phos.
ID	Lot ID	(v:v)	Viability	(cells/cc)	Staining
A	1	5:2	85.5%	2.761,500	Positive
B	1	2:1	86.3%	2,732,750	Positive
C	2	5:2	87.8%	2,824,750	Positive
D	2	2:1	89.0%	2,767,000	Positive

Claims

1. A bone graft composition comprising: bone particles containing viable osteogenic cellular material native to the bone particles;a viscous cryoprotectant comprising at least one penetrating cryoprotective agent and at least one non-penetrating cryoprotective agent, wherein the at least one non-penetrating cryoprotective agent comprises alginate,wherein the viscous cryoprotectant comprises a concentration of alginate of 4% to 6%; anda demineralized bone matrix,wherein a volume ratio of the bone particles to the demineralized bone matrix is in a range from 5:2 to 53, and wherein the bone graft composition is homogenous and malleable,wherein a second volume ratio of the bone particles to the viscous cryoprotectant is from 5:2 to 2:1, andwherein at least seventy percent of the viable osteogenic cellular material is viable after storage in the viscous cryoprotectant at −80 degrees Celsius or lower for a period of fourteen days.

2. The bone graft composition of claim 1, wherein the bone particles are from viable cancellous bone, and the demineralized bone matrix is from cortical bone.

3. The bone graft composition of claim 1, wherein the bone particles are cohesively bounded by the viscous cryoprotectant.

4. The bone graft composition of claim 1, wherein the bone particles are coated or encapsulated by the viscous cryoprotectant.

5. The bone graft composition of claim 1, wherein a viscosity of the viscous cryoprotectant is higher than 2000 centipoises (cps).

6. The bone graft composition of claim 1, wherein the non-penetrating cryoprotective agent further comprises one or more of hyaluronic acid, hydroxyethyl starch, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, chitosan, and glycerol.

7. The bone graft composition of claim 1, wherein the penetrating cryoprotective agent is one of dimethyl sulfoxide, glycerol, propylene glycol, ethylene glycol, propanediol, or a combination thereof.

8. A method of preserving viability of a bone graft material, the method comprising: combining viable bone graft material with a demineralized bone matrix, wherein a volume ratio of the viable bone graft material to the demineralized bone matrix ranges from 5:2 to 5:3 and wherein the viable bone graft material contains viable osteogenic cells inherent thereto; andcombining the viable bone graft material and the demineralized bone matrix with a viscous cryoprotectant into a homogenous composition for preserving the viability of the viable bone graft material, wherein the viscous cryoprotectant includes at least one non-penetrating cryoprotective agent and at least one penetrating cryoprotective agent, wherein the at least one non-penetrating cryoprotective agent comprises alginate,wherein a second volume ratio of the viable bone graft material to the viscous cryoprotectant is from 5:2 to 2:1,wherein the viscous cryoprotectant comprises a concentration of alginate of 4% to 6%, and wherein at least seventy percent of the viable osteogenic cellular material is viable after storage in the viscous cryoprotectant at −80 degrees Celsius or lower for a period of fourteen days.

9. The method claim 8, wherein the viable bone graft material is from viable cancellous bone, and the demineralized bone matrix is from viable cortical bone.

10. The method claim of 8, wherein the viable bone graft material is from viable cortical bone, and the demineralized bone matrix is from viable cancellous bone.

11. The method claim 8, wherein the viable bone graft material is cohesively bounded by the viscous cryoprotectant.

12. The method of claim 8, wherein a viscosity of the viscous cryoprotectant is higher than 2000 centipoises (cps).

13. The method claim of 8, wherein the non-penetrating cryoprotective agent further comprises one or more of hyaluronic acid, hydroxyethyl starch, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, chitosan, and glycerol.

14. The method claim of 8, wherein the penetrating cryoprotective agent is one of dimethyl sulfoxide, glycerol, propylene glycol, ethylene glycol, propanediol, or a combination thereof.