**4. Discussion**

Demineralized bone matrices (DBMs) are widely used in spinal, orthopedic, craniomaxillofacial, and dental procedures to treat bone voids. An ideal DBM provides both osteoinductive and osteoconductive properties to promote new bone formation and provide a scaffold upon which cells can attach and proliferate. Furthermore, DBMs should be malleable and resist graft migration once impacted into a bone defect. To achieve these characteristics important for bone healing, manufacturers use a variety of techniques to process and sterilize DBMs. Despite demineralization being a well-known technique, the proportion of the osteoinductive element—the demineralized bone—of clinically available DBM-based graft materials varies widely by manufacturer. Differences in carrier material and sterilization may also contribute to variability among these grafts. The moldable demineralized fibers described here represent a recently developed allograft configuration that can function as an independent bone void filler without the need of a synthetic carrier. This study was conducted to ensure L-MDF possess the necessary qualities to function in this capacity.

An osteoinductive bone graft has the ability to induce bone growth. Factors such as residual calcium level and growth factor content play important roles in a DBM's ability to grow bone. In particular, residual calcium level can serve as an indicator for the availability of growth factors necessary for bone formation. The literature suggests that DBMs with different degrees of residual calcium show significant differences in osteoinductivity. Zhang et al. evaluated the effects of varying degrees of demineralization, particle size, donor age, and gender on the osteoinductivity of DBM *in vivo* (athymic mouse model) and *in vitro* (alkaline phosphatase assay) [22]. The authors suggested that demineralized bone with a residual calcium level of approximately 2% is "optimally osteoinductive". Similarly, Turonis et al. found that a 2% residual calcium level in human demineralized freeze-dried bone allograft appears to enhance osseous wound healing [23]. The L-MDF samples discussed in this chapter were demineralized using a proprietary and patented process targeted at achieving an optimized level of residual calcium of 1–4%. Furthermore, the presence of specific proteins in DBM is frequently associated with its osteoinductive potential as growth factors can provide signals that direct cellular behavior [18, 22, 24]. In particular, BMP-2 and 7 are important for bone growth as they are known for their "ability to stimulate differentiation of MSCs to osteochondroblastic lineage" [18]. Previous studies have reported a wide span of BMP-2 and BMP-7 levels in demineralized bone, with ranges from 6.5 to 110 and 44 to 125 ng/g demineralized bone, respectively. In this study, ELISA results indicated the presence of BMP-2 and 7 in L-MDF (11.24 ± 1.49 and 85.78 ± 6.84 ng/g) consistent with values reported in the literature. This milieu of growth factors illustrate that L-MDF contain the appropriate trophic factor profile necessary for bone formation and are consistent with expected physiological levels.

The osteoinductive and osteoconductive potential of DBMs are commonly evaluated using an *in vivo* athymic mouse intramuscular pouch model to histologically assess new bone formation [25].

In the study described here, histological analysis revealed the presence of new bone elements demonstrating the osteoinductive potential of L-MDF. In addition, newly formed blood vessels were observed, which can also be indicative of the

**41**

to an SAL of 10<sup>−</sup><sup>6</sup>

the results presented here.

*Cell Attachment and Osteoinductive Properties of Tissue Engineered, Demineralized Bone Fibers…*

osteoconductive nature of the bone graft in providing a conducive environment for new bone formation. The surface characteristics of DBMs play an important role in their ability to provide a scaffold for new bone formation [19, 20]. Bone cells need a hospitable environment in which to attach and thrive. In particular, increased surface area, a rough topography, and interconnected networks are known to promote cellular attachment and cell spreading [26]. As demonstrated by the SEM imaging presented here, the long, interconnected L-MDF create a hospitable environment for BM-MSCs to infiltrate and make cell-to-cell connections. The ability of cells not only to quickly attach to the matrix but also maintain a healthy morphology throughout the duration of culture provides evidence of the osteoconductive quali-

The need for versatile handling has led to the addition of various inert carriers in commercial DBMs. However, studies have shown that carriers may negatively affect the inherent properties of a DBM. In particular, Lee et al. concluded that Poloxamer 407-based hydrogel may inhibit MSC osteoblastic differentiation by filling up spaces between DBM powders, negatively affecting the release of growth factors [21]. In a rat calvarial defect model, investigators found that the two types of DBM had significant differences in bone regeneration, which was attributed to the type of carrier [27]. Furthermore, varying the ratio of carrier to DBM can alter handling characteristics such as malleability and resistance to graft migration. Through *in vivo* and *in vitro* analyses, studies have found that increased bone content in DBMs produces larger amounts of new bone formation [25, 28, 29]. With this is mind, L-MDF were produced by proprietary CNC-milling cortical bone to create specially designed rough surfaces allowing fibers to interlock, allowing this bone void filler to be carrier-free. The roughness also provides numerous attachment points for the cells and their lamellipodia, encouraging a flattened morphology. These interlocking fibers thereby encourage malleability, graft placement in the implant site, and resistance to irrigation, all of which represent ideal handling

Finally, terminal sterilization is a processing measure used to ensure the safety of DBMs by reducing the risk of disease transmission. This is in contrast to aseptic processing alone, which introduces no additional bioburden from the environment but alone does not guarantee sterile tissue [30, 31]. Unlike aseptic-only processed tissue, terminal sterilization can result in a graft with a defined sterility assurance

a viable organism exists within any single graft [31]. Although gamma irradiation is currently the most common method for terminally sterilizing allografts, some reports suggests that gamma irradiation can negatively impact the inherent properties of DBMs. There are several factors to consider when evaluating the effects of gamma irradiation on DBMs such as dose and temperature. Irradiation performed in a high dose range or at uncontrolled temperatures can result in denaturing of the osteoinductive signaling proteins, rendering them inactive, and/or structural damage to the collagen matrix due to generation of reactive oxygen species. Weintroub and Reddi evaluated DBM samples which were irradiated on ice at varying doses [32]. Histologic analysis showed DBM irradiated at 0.5–2.5 Mrad were similar to the non-irradiated control, indicating no effect on the induction properties of the implant. In another study, investigators found that DBM irradiated on dry ice (−72°C) demonstrated new bone formation comparable to non-irradiated samples [33]. These results demonstrate DBMs irradiated at low dose and low temperatures are expected to retain properties important to clinical performance. Thus, L-MDF are terminally sterilized

negative impacts to the osteoinductive and osteoconductive potential, as verified by

using low-dose, ultra-low temperature gamma irradiation to avoid

indicates a 1 out of 1,000,000 chance that

*DOI: http://dx.doi.org/10.5772/intechopen.88290*

ties of L-MDF.

characteristics.

level (SAL). For example, an SAL of 10<sup>−</sup><sup>6</sup>

#### *Cell Attachment and Osteoinductive Properties of Tissue Engineered, Demineralized Bone Fibers… DOI: http://dx.doi.org/10.5772/intechopen.88290*

osteoconductive nature of the bone graft in providing a conducive environment for new bone formation. The surface characteristics of DBMs play an important role in their ability to provide a scaffold for new bone formation [19, 20]. Bone cells need a hospitable environment in which to attach and thrive. In particular, increased surface area, a rough topography, and interconnected networks are known to promote cellular attachment and cell spreading [26]. As demonstrated by the SEM imaging presented here, the long, interconnected L-MDF create a hospitable environment for BM-MSCs to infiltrate and make cell-to-cell connections. The ability of cells not only to quickly attach to the matrix but also maintain a healthy morphology throughout the duration of culture provides evidence of the osteoconductive qualities of L-MDF.

The need for versatile handling has led to the addition of various inert carriers in commercial DBMs. However, studies have shown that carriers may negatively affect the inherent properties of a DBM. In particular, Lee et al. concluded that Poloxamer 407-based hydrogel may inhibit MSC osteoblastic differentiation by filling up spaces between DBM powders, negatively affecting the release of growth factors [21]. In a rat calvarial defect model, investigators found that the two types of DBM had significant differences in bone regeneration, which was attributed to the type of carrier [27]. Furthermore, varying the ratio of carrier to DBM can alter handling characteristics such as malleability and resistance to graft migration. Through *in vivo* and *in vitro* analyses, studies have found that increased bone content in DBMs produces larger amounts of new bone formation [25, 28, 29]. With this is mind, L-MDF were produced by proprietary CNC-milling cortical bone to create specially designed rough surfaces allowing fibers to interlock, allowing this bone void filler to be carrier-free. The roughness also provides numerous attachment points for the cells and their lamellipodia, encouraging a flattened morphology. These interlocking fibers thereby encourage malleability, graft placement in the implant site, and resistance to irrigation, all of which represent ideal handling characteristics.

Finally, terminal sterilization is a processing measure used to ensure the safety of DBMs by reducing the risk of disease transmission. This is in contrast to aseptic processing alone, which introduces no additional bioburden from the environment but alone does not guarantee sterile tissue [30, 31]. Unlike aseptic-only processed tissue, terminal sterilization can result in a graft with a defined sterility assurance level (SAL). For example, an SAL of 10<sup>−</sup><sup>6</sup> indicates a 1 out of 1,000,000 chance that a viable organism exists within any single graft [31]. Although gamma irradiation is currently the most common method for terminally sterilizing allografts, some reports suggests that gamma irradiation can negatively impact the inherent properties of DBMs. There are several factors to consider when evaluating the effects of gamma irradiation on DBMs such as dose and temperature. Irradiation performed in a high dose range or at uncontrolled temperatures can result in denaturing of the osteoinductive signaling proteins, rendering them inactive, and/or structural damage to the collagen matrix due to generation of reactive oxygen species. Weintroub and Reddi evaluated DBM samples which were irradiated on ice at varying doses [32]. Histologic analysis showed DBM irradiated at 0.5–2.5 Mrad were similar to the non-irradiated control, indicating no effect on the induction properties of the implant. In another study, investigators found that DBM irradiated on dry ice (−72°C) demonstrated new bone formation comparable to non-irradiated samples [33]. These results demonstrate DBMs irradiated at low dose and low temperatures are expected to retain properties important to clinical performance. Thus, L-MDF are terminally sterilized to an SAL of 10<sup>−</sup><sup>6</sup> using low-dose, ultra-low temperature gamma irradiation to avoid negative impacts to the osteoinductive and osteoconductive potential, as verified by the results presented here.

*Clinical Implementation of Bone Regeneration and Maintenance*

marrow, new blood vessels, and new bone.

**4. Discussion**

to function in this capacity.

consistent with expected physiological levels.

cally assess new bone formation [25].

new bone elements present in the explant (4× objective). Panels B and C highlight the presence of new bone elements such as cartilage, chondroblasts/cytes, bone

Demineralized bone matrices (DBMs) are widely used in spinal, orthopedic, craniomaxillofacial, and dental procedures to treat bone voids. An ideal DBM provides both osteoinductive and osteoconductive properties to promote new bone formation and provide a scaffold upon which cells can attach and proliferate. Furthermore, DBMs should be malleable and resist graft migration once impacted into a bone defect. To achieve these characteristics important for bone healing, manufacturers use a variety of techniques to process and sterilize DBMs. Despite demineralization being a well-known technique, the proportion of the osteoinductive element—the demineralized bone—of clinically available DBM-based graft materials varies widely by manufacturer. Differences in carrier material and sterilization may also contribute to variability among these grafts. The moldable demineralized fibers described here represent a recently developed allograft configuration that can function as an independent bone void filler without the need of a synthetic carrier. This study was conducted to ensure L-MDF possess the necessary qualities

An osteoinductive bone graft has the ability to induce bone growth. Factors such as residual calcium level and growth factor content play important roles in a DBM's ability to grow bone. In particular, residual calcium level can serve as an indicator for the availability of growth factors necessary for bone formation. The literature suggests that DBMs with different degrees of residual calcium show significant differences in osteoinductivity. Zhang et al. evaluated the effects of varying degrees of demineralization, particle size, donor age, and gender on the osteoinductivity of DBM *in vivo* (athymic mouse model) and *in vitro* (alkaline phosphatase assay) [22]. The authors suggested that demineralized bone with a residual calcium level of approximately 2% is "optimally osteoinductive". Similarly, Turonis et al. found that a 2% residual calcium level in human demineralized freeze-dried bone allograft appears to enhance osseous wound healing [23]. The L-MDF samples discussed in this chapter were demineralized using a proprietary and patented process targeted at achieving an optimized level of residual calcium of 1–4%. Furthermore, the presence of specific proteins in DBM is frequently associated with its osteoinductive potential as growth factors can provide signals that direct cellular behavior [18, 22, 24]. In particular, BMP-2 and 7 are important for bone growth as they are known for their "ability to stimulate differentiation of MSCs to osteochondroblastic lineage" [18]. Previous studies have reported a wide span of BMP-2 and BMP-7 levels in demineralized bone, with ranges from 6.5 to 110 and 44 to 125 ng/g demineralized bone, respectively. In this study, ELISA results indicated the presence of BMP-2 and 7 in L-MDF (11.24 ± 1.49 and 85.78 ± 6.84 ng/g) consistent with values reported in the literature. This milieu of growth factors illustrate that L-MDF contain the appropriate trophic factor profile necessary for bone formation and are

The osteoinductive and osteoconductive potential of DBMs are commonly evaluated using an *in vivo* athymic mouse intramuscular pouch model to histologi-

In the study described here, histological analysis revealed the presence of new bone elements demonstrating the osteoinductive potential of L-MDF. In addition, newly formed blood vessels were observed, which can also be indicative of the

**40**
