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

PRIORITY CLAIM

This application is a continuation of, and claims priority to U.S. patent application Ser. No. 16/995,128 (filed Aug. 17, 2020), which itself claims priority to U.S. Pat. No. 10,780,197 (U.S. patent application Ser. No. 14/066,589, filed Oct. 29, 2013), which claims priority to U.S. Provisional Patent Application No. 61/719,868 (filed Oct. 29, 2012), each of which is incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure generally relates to medical devices. More particularly, the disclosure relates to the field of spinal surgery and spinal fixation devices.

BACKGROUND

The spine is critical in human physiology for mobility, support, and balance. Spinal injuries can be debilitating or catastrophic to patients. Even small irregularities in the spine can cause devastating pain and loss of coordination.

Surgical procedures are commonly performed to correct problems with displaced, damaged, or degenerated intervertebral discs. Generally, spinal fusion procedures involve removing some or all of the diseased or damaged disc, and inserting one or more intervertebral implants into the resulting disc space. Successful replacement of injured or deteriorated spinal bone with artificial implants can involve consideration and understanding of the inherent stresses on the spine, as well as the biological properties of the body in response to the devices.

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.

Two or more features described in this disclosure, including those described in this summary section, may be combined to form implementations not specifically described herein.

The above presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key or critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features, objects and benefits will be apparent from the description and drawings, and from the claims.

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 um. Cortical bone was ground, sieved to 125-1000 um, 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 um. Cortical bone was ground, sieved to 100-710 um, 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.

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 um. Cortical bone was ground, sieved to 100-710 um, 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.

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 um. Cortical bone was ground, sieved to 125-1000 um, 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 um. Cortical bone was ground, sieved to 125-1000 um, 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
	DMSO	Human Serum		Avg.
Compo-	Concen-	Albumin	Avg.	Cell
sition	tration	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 um. Cortical bone was ground, sieved to 125-1000 um, 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.

TABLE 8
				Avg.
Compo-				Cell	Alk.
sition	Alginate	Cancellous:Cryo	Avg. 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

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. As used in this specification and the claims, unless otherwise stated, the term “about,” “approximately,” “generally,” and “substantially” refers to variations of less than or equal to +/−1%, +/−2%, +/−3%, +/−4%, +/−5%, +/−6%, +/−7%, +/−8%, +/−9%, +/−10%, +/−11%, +/−12%, +/−14%, +/−15%, +/−16%, +/−17%, +/−18%, +/−19%, or +/−20%, depending on the embodiment. As a further non-limiting example, about 100 millimeters represents a range of 95 millimeters to 105 millimeters, 90 millimeters to 110 millimeters, or 85 millimeters to 115 millimeters, depending on the embodiments.

While inventive features described herein have been described in terms of preferred embodiments for achieving the objectives, it will be appreciated by those skilled in the art that variations may be accomplished in view of these teachings without deviating from the spirit or scope of the invention. Also, while this invention has been described according to a preferred use in spinal applications, it will be appreciated that it may be applied to various other uses desiring surgical fixation, for example, the fixation of long bones.

A number of implementations have been described. Nevertheless, it will be understood that additional modifications may be made without departing from the scope of the inventive concepts described herein, and, accordingly, other implementations are within the scope of the following claims.

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 at least 4%; and

a demineralized bone matrix,

wherein the bone graft composition is homogenous and malleable.

2. The bone graft composition of claim 1, wherein a first volume ratio of the bone particles to the demineralized bone matrix is in a range from 5:2 to 5:3.

3. The bone graft composition of claim 2, wherein a second volume ratio of the bone particles to the viscous cryoprotectant is from 5:2 to 2:1.

4. 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.

5. The bone graft composition of claim 1, wherein at least seventy-four percent of the viable osteogenic cellular material is viable after storage in the cryopreservative at −80 degrees Celsius or lower for a period of fourteen days.

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

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

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

9. The bone graft composition of claim 1, wherein the at least one non-penetrating cryoprotective agent further comprises one or more of hyaluronic acid, hydroxyethyl starch, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, chitosan, and glycerol, and wherein the at least one penetrating cryoprotective agent is one of dimethyl sulfoxide, glycerol, propylene glycol, ethylene glycol, propanediol, or a combination thereof.

10. The bone graft composition of claim 1, wherein the viscous cryoprotectant comprises a concentration of alginate of at least 6%, and wherein at least eighty-five percent of the viable osteogenic cellular material is viable after storage in the cryopreservative at −80 degrees Celsius or lower for a period of fourteen days.

11. A method of preserving viability of a bone graft composition, the method comprising:

combining a viable bone graft material with a demineralized bone matrix, wherein the viable bone graft material contains viable osteogenic cells inherent thereto; and

combining 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, and wherein the viscous cryoprotectant comprises a concentration of alginate of at least 4%.

12. The method claim of 11, wherein a first volume ratio of the viable bone graft to the demineralized bone matrix ranges from 5:2 to 5:3, and a second volume ratio of the viable bone graft material to the viscous cryoprotectant is from 5:2 to 2:1.

13. The method claim of 11, wherein:

a) the viable bone graft material is from viable cancellous bone, and the demineralized bone matrix is from viable cortical bone,

b) the viable bone graft material is from viable cortical bone, and the demineralized bone matrix is from viable cancellous bone, or

c) the viable bone graft material is cohesively bounded by the viscous cryoprotectant.

14. The method claim of 11, wherein at least seventy-four percent of the viable osteogenic cells are viable after storage in the cryopreservative at −80 degrees Celsius or lower for a period of fourteen days.

15. The method claim of 11, wherein the viable bone graft material is coated or encapsulated by the viscous cryoprotectant, wherein the viscous cryoprotectant comprises a concentration of alginate of at least 6%, and wherein at least eighty-five percent of the viable osteogenic cellular material is viable after storage in the cryopreservative at −80 degrees Celsius or lower for a period of fourteen days.

16. The method claim of 11, wherein a viscosity of the viscous cryoprotectant is higher than 2000 centipoises (cps).

17. The method claim of 11, wherein the at least one non-penetrating cryoprotective agent further comprises one or more of hyaluronic acid, hydroxyethyl starch, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, chitosan, and glycerol.

18. The method claim of 11, wherein the at least one penetrating cryoprotective agent is one of dimethyl sulfoxide, glycerol, propylene glycol, ethylene glycol, propanediol, or a combination thereof.

19. 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; and

a demineralized bone matrix,

wherein the bone graft composition is homogenous and malleable,

wherein at least seventy-four percent of the viable osteogenic cellular material is viable after storage in the cryopreservative at −80 degrees Celsius or lower for a period of fourteen days.

20. The bone graft composition of claim 19, wherein at least eighty percent of the viable osteogenic cellular material is viable after storage in the cryopreservative at −80 degrees Celsius or lower for the period of fourteen days.