American Association of Plastic Surgeons – Annual Meeting
Accepted Research Abstract Presented March 23, 2009
Porcine Allograft Mandible Revitalization Using Osteogenically-Induced Autologous Adipocyte-Derived Stem Cells and Periosteum
Jesse A. Taylor, MD, Donna C. Jones, PhD, Rian A. Maercks, MD, Christopher B. Gordon, MD, David A. Billmire, MD, Christopher M. Runyan, PhD.
Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
Purpose
Critical defects of the craniomaxillofacial region, as well as long bones, often are treated with vascularized osteocutaneous free flaps. These are lengthy operations, may be associated with considerable donor site morbidity, and often have suboptimal results, both functionally and aesthetically. The prospect of tissue engineering vascularized bone offers an attractive alternative.
The purpose of this study was to engineer a vascularized bone flap that approximates the pig hemi-mandible using acellular bone allograft, adipose-derived mesenchymal stem cells, and recombinant human BMP-2. Our experiment investigated the contribution of a vascular pedicle within the center of the construct and a complete vascularized periosteal envelope.
Methods
Edentulous allograft porcine mandibles were separated at the symphysis and commercially Biocleansed (RTI) to remove all antigenic material. Adipocyte-derived mesenchymal stem cells (ASCs) were harvested from 10 pigs using liposuction, expanded in culture, and autogenously implanted into the allografts. The allografts were then surgically placed into one of two locations within the pig: 1) a complete periosteal envelope supplied by two adjacent intercostal vessels after extraction of two ribs (‘thoracic’), 2) wrapped within the rectus abdominis muscle after insertion of the superficial inferior epigastric vascular pedicle into the medullary cavity (‘abdominal’). In each model, BMP-2-soaked collagen-I sponge and ASCs were placed into the medullary space of the allograft. The constructs were allowed to incubate in vivo for 7-8 weeks, and then harvested to assess de novo bone formation using imaging studies and histology.
Results
Micro-CT scans (100 μm slices) of each harvested implant were examined and calcitic tissue was quantified utilizing the gray scale X-ray attenuation coefficient and thee dimensional reconstructions of the bony versus soft tissue. Abdominal implants contained 143.20 (± 46.39) mL of calcitic tissue, while thoracic implants had 474.16 (± 75.93) mL. ANOVA demonstrated that thoracic implants had significantly more calcitic tissue than did abdominal ones (p < 0.006). Histologic analysis of abdominal allografts showed minimal new (woven) cancellous bone, indicating the calcitic tissue present was that of the implant. In contrast, thoracic allografts were almost entirely composed of extensive new (woven) cancellous bone and most of the implant had been absorbed. The thoracic allografts demonstrated Haversian systems, marrow elements and blood vessels resembling normal bone whereas abdominal allografts did not.
Conclusion
These data are the first to demonstrate revitalization of large volume allograft bone, and have positive implications for the tissue-engineering of the craniomaxillofacial and axial skeleton. The massive growth of bone in the thoracic but not abdominal allograft suggests that periosteum is critical in the formation of new bone. Further studies are ongoing to determine the exact role of periosteum and the interplay of ASCs and BMP-2 in the process.
A quick glance at engineering vascularized bone:
From the Lab to the Operating Suite
The prospect of tissue engineering vascularized bone is an attractive and needed alternative to current methods for repairing craniofacial and long-bone defects caused by disease or traumatic injury. Growing new bone to replace damaged or missing bone has long been pursued by medical science with mixed success. Scientists and surgeons at Cincinnati Children’s have combined and refined techniques under study or already used in reconstructive surgery to find that better solution.
The need is critical. An estimated 7 million people in the United States have defects in bone continuity so severe that repair is difficult. Current methods – such as borrowing bone from another part of the body, implanting cadaver bone or an artificial component – are less than ideal. Failure rates for these procedures can be high because the body either rejects or absorbs the implanted material. This can cause high patient morbidity and require repeated procedures.
Success in the Laboratory
Results prepared for peer-review journal publication and presented at medical conferences show scientists at Cincinnati Children’s have been able to engineer vascularized mandible (jaw) bone in pigs. The experiments are the first to demonstrate successful revitalization of a large volume of transplanted cadaver bone (called an allograft), with positive implications for tissue engineering bones of the face, skull and thoracic regions. The porcine immune system, similar to the human system, makes pigs a good model for simulating human bone growth.
Researchers used bio-cleansed mandible bones from pig cadavers as allograft implants. They harvested fat-derived mesenchymal stem cells from living pigs and expanded the cell populations in laboratory cultures. These non-embryonic stem cells can become cells for many, but not all, tissues types in the body – including connective tissue and bone. The allograft bones were infused with the stem cells and an engineered synthetic form of a growth protein that stimulates bone growth – bone morphogenic protein-2. BMP-2 cues the stem cells to form bone cells. Once that process starts, BMP-2 is produced naturally by the body.
Prepared bone allografts were surgically implanted in the thoracic regions of the pigs and surrounded by periosteum envelopes, supplied by adjacent blood vessels. Periosteum is critical to success as it provides vascularization and a nurturing blood supply to rejuvenating bone.
The bone implants were incubated in the pigs’ thoracic regions for eight weeks then removed for analysis, revealing extensive formation of new bone. The new bone showed elements of marrow and blood vessels, similar to normal bone.
Translating Science into Successful Treatment
In the first procedure of its kind, reconstructive surgeons at Cincinnati Children’s translated the laboratory findings into a successful attempt to grow complex cheek bones (known as the zygomatic) in the face of a 14-year-old boy. The active teen was born without fully developed cheek bones, a critical protective feature, putting his eyes at risk of injury.
Using cadaver bone allografts as implants – which essentially served as growth guides and scaffolding for new bone tissue – the surgeons harvested mesenchymal stem cells from the teenager’s abdominal fat. During the day-long procedure in May 2009, the allografts were implanted into the teen’s face with surgical screws and the shaped donor bone was infused with the harvested stem cells and BMP-2. Surgeons used tissue from the teen’s thigh as periosteum to wrap the implants and provide blood supply.
Four months post surgery, CT scans and physical examination showed the allografts had successfully rejuvenated into dense facial bone with no sign of rejection or absorption. A key advantage to avoiding rejection is that the stem cells and periosteum come from the implant recipient’s own body.
