(192h) Advanced Fracture Healing with Hemicellulose Polymer

PURPOSE:  The purpose of this research was to improve the healing of large bone fractures at risk of delayed healing becoming a non-union.  Plants that do not produce the hemicellulose polymer Xylan have weak stems and cannot bear weight.  Xylan regulates cellulose fiber diameter and orientation in plant secondary cell walls, resulting in a stronger structure of parallel fibers rather than a disorganized aggregation of fibers.  If Xylan can similarly regulate collagen fiber formation during bone repair, combining Xylan with the antibacterial and gelation properties of chitosan would result in a versatile tool in the treatment of bone injuries.

DESIGN:  The hypothesis is that injection of a hydrogel composed of xylan and chitosan will improve healing of large bone fractures.  In vivomodels were ectopic implants in mice and rats, tibia fractures in mice, and femur fractures in rats.  The fracture models used 5 animals in each study group.  The subcutaneous models used 3 animals in each group with two implants in each animal for a total of 6 samples for each group.

METHODS:  Xylan and chitosan associate with each other in solution without further aid and form a composite hydrogel using the same process as previously reported chitosan thermo-responsive gels.  The xylan/chitosan composite hydrogel and pure chitosan control were injected under the skin on the back of each mouse.  The two hydrogels were also loaded onto sintered microsphere scaffolds and implanted in the rat thigh.  After one week all implants were removed for histological staining.  Mouse tibias were pinned with a steel rod, fractured by blunt trauma, and treated with injections of pure chitosan or the xylan/chitosan hydrogels directly into the fractured area.  Rat femurs were fractured in the same manner and received the same treatments.  X-ray images were taken of fractured bones once per week to monitor the progression of healing.

FINDINGS:  In the subcutaneous model, the xylan/chitosan composite implant was completely replaced with vascularized host tissue while the chitosan implant was still evident at the injection site.  In the rat thigh muscle, host tissue penetrated to the center of scaffolds loaded with composite hydrogel, but pure chitosan acted as a barrier with no penetration of host tissue.  Advanced healing of mouse tibia fractures was evident by the rapid remodeling of fracture callus to normal bone between three and four weeks while callus remained in other groups.  Rat femur fractures showed consistent and early callus formation at two weeks in response to the xylan/chitosan composite compared to wide variation in response to other treatment groups, from no healing to large callus overgrowth in both control and pure chitosan groups.

CONCLUSIONS:  The xylan/chitosan composite hydrogel demonstrated improved healing responses even without addition of therapeutic cells or growth factors.  Treatment with this new material consistently improved fracture healing in two animal models and promoted tissue growth in two ectopic models.

IMPLICATIONS:  Successful application of xylan to fracture healing reveals a new class of hemicellulose polymers not yet applied in this field.  Because of xylan’s role in regulating fiber size and orientation, the impact of this study may have a broader impact on a wide range of injuries and diseases that are prone to fibrosis.

FUNDING:  DOD Hypothesis Development Award (W81XWH-10-1-0886) and SCV R. Clifton Brooks Jr. Medical Research Fellowship.