Periosteum plays a critical role in bony healing. Eyre-Brook1 described the role of the periosteum in fracture or bone loss situations as providing a link between fragments of bone and functioning as a revascularization conduit to unite a fracture. Periosteum facilitates nutrient supply and delineates a cavity to maintain the hematoma that aids the remaining callus edges to bridge.1 The loss of the periosteal link and hematoma during healing may result in fibrous or delayed union of the bony fragment.1
Macnab and De Haas2 researched the importance of the periosteum as it relates to the infiltration of fibrous tissue between bony fragments. Thenstudy demonstrated that an intact periosteal seal can help prevent a fibrous union between the ends of bone because the seal minimizes fibrous tissue invasion from damaged soft tissue.2
Grundnes and Reikeras3 showed the importance of the periosteal seal in preventing the escape of the local hematoma and mesenchymal cells into the local soft tissue.
Surgical techniques that maintain intact periosteum should be used for repair of bone defect sites, but there are clinical situations when damaged or inadequate periosteum does not allow for intact autologous periosteum to be used in conjunction with bone repair.
There are many attributes to consider when developing a periosteum replacement scaffold intended to mimic the characteristics of autologous periosteum. Important attributes include maintenance of the underlying bone site and osteogenic cells, facilitation of nutritional diffusion, selective exclusion of competing muscle and soft tissue cell invasion, barrier provision for fibrous and other nonosseous tissue ingrowth, and retention and containment of the medullary canal.
GraftJacket Acellular Periosteum Replacement Scaffold (Wright Medical Technology, Ine, Arlington, Tenn) may provide biochemical and physical attributes that act as a template for the repair of damaged or inadequate periosteum in bone repair sites.
The scaffold is composed of donated human-derived dermal tissue that contains critical extracellular matrix components such as collagen, elastin, and proteoglycans. The tissue has been processed to remove all cellular components while maintaining its biochemical matrix. Patented cryogenic processing is used to preserve the extracellular matrix, including vascular channels.
Studies with the acellular dermal matrix (LifeCell, Branchburg, NJ) have shown that the material supports neovascularization and fibroblast infiltration and incorporates into host tissue with no cell-mediated immune responses or inflammatory cell infiltration.4
The dermal matrix has been used in clinical applications that include urological slings, skin grafts, soft tissue augmentation, and dental barrier membrane applications.59 A study using the acellular dermal matrix for guided bone regeneration in the staged placement of a dental implant indicated that the material allowed for bone regeneration and uneventful healing.9
The preclinical data presented in this article demonstrate that the decellularized dermal material allows for cell repopulation and revascularization, provides a protected environment for bone defect restoration, and minimizes fibrous tissue invasion that encompasses many of the important attributes needed for periosteum replacement.
MATERIALS AND METHODS
Athymic Nude Rat Muscle Implantation Study
A submuscular implantation of the human acellular dermal matrix in athymic nude rats was implemented to evaluate the cellular repopulation and revascularization of the material over time. The use of athymic rats allowed human dermal material to be tested without the potential for an immunogenic reaction that can be evoked when human-derived material is used in nonhuman species.
Male athymic nude rats were anesthetized with an intramuscular (IM) injection of ketamine hydrochloride (87 mg/kg) and xylazine (13 mg/kg). Isofluorane (0.5%-5%; with an O2 carrier) was used to maintain anesthesia during procedures.
A 3-cm incision through the skin was created in the mid-dorsum of the athymic nude rat. Incisions 1.5 cm in size were made through the latissimus dorsi on each side of the midline, and two pouches (both approximately 1 cm2) were created on each side using blunt dissection. Humanderived acellular dermal sheets were rehydrated in saline for approximately 10 minutes. Specimens were then removed from the saline and cut into 1 cm2 specimens.
The specimens were implanted bilaterally in the muscle pouches. Each specimen was placed flat in the pouch and pushed away from the muscle incision. After implantation, the muscle incisions were closed with an interrupted 4-0 Vicryl suture (Ethicon, Ine, Somerville, NJ), and the skin incision was closed with an interrupted 4-0 Ethicon suture (Ethicon, Ine). The time points for sample explantation were at 2, 4, 7, 14, and 21 days after implantation (N=2 per time point).
Animals were sacrificed with an overdose of sodium pentobarbitol euthanasia solution. After each tissue capsule was excised, specimens were stored in neutral buffered formalin for 24 to 48 hours after extraction and then transferred to isopropyl alcohol until processing.
Each specimen was cut into 2-3 pieces (cross-sections) and embedded in paraffin. Cut edges from one specimen were placed on a slide, with two slides per specimen. Hematoxylin and eosin (H&E) was used to elucidate cells and nuclei. Slides were examined for extent of cellular infiltration and vascularity.
Porcine Femoral Drill Hole Study
A porcine femoral drill hole defect study was conducted using acellular dermal porcine membrane to assess cellular repopulation, revascularization, and periosteum scaffold characteristics. Porcine decellularized matrix was used in a porcine animal model because of the potential for an immunogenic reaction when human-derived material is used in nonhuman species.
Adult Yucatan mini-pigs were anesthetized with ketamine HCl/zolazepam HCl 8 mg/kg, ketamine 4 mg/kg, and xylazine 4 mg/kg. They were intubated and maintained on 2%-3% isoflurane and 1 -2 L of O2/minute.
Preoperative medications included approximately 40 mg/kg cefazolin intravenously, 0.007 mg/kg buprenorphine intramuscularly, and 0.01 mg/kg glycopyrrolate intramuscularly. Postoperative analgesia included 0.007 mg/kg buprenormorphine intramuscularly and 50 ug/hour fentanyl (transdermal) patches placed every 1 to 3 days as needed.
Drill hole defects 3/8 inch in diameter and 5 mm in depth were made in the femoral shafts. The drill hole sites were filled with various test materials (N=I), including porcine demineralized bone matrix (DBM) and morsellized autologous bone.
The porcine decellularized dermal material was rehydrated in saline for approximately 10 minutes. The scaffold sheets were cut to overlap the defect site by approximately 2-4 mm. The porcine dermal membranes were adhered to the site with Tisseel (Baxter, Glendale, Calif) fibrin glue and surgical tacks or Nexaband (Closure Medical Corp, Raleigh, NC) veterinary cyanoacrylate glue.
Fascial and subcutaneous tissue was closed with a simple continuous 3-0 PDS suture (Ethicon, Ine). The femoral skin was closed with a 2-0 Dermalon (United States Surgical AutoSuture and USS/DG, Divisions of Tyco Healthcare Group LP, Norwalk, Conn) cruciate pattern and the tibial skin was closed with staples.
Figure 1: Cellular repopulation at day 2 (x20 magnification) (A), day 4 (x20 magnification) (8), and day 7 (x20 magnification) (Q. Cellular repopulation and revascularization at day 14 (X20 magnification) (D) and day 21 (x20 magnification) (E).
At 5 weeks, the animals were humanely euthanized with a captive bolt and exsanguinated, and the femora were isolated. Histological analysis was conducted at the conclusion of the study. Routine H&E staining was performed on cross-sections of the defects. Factor VHI-related antigen immunostaining of the porcine defects was also performed to better identify areas of neovasculature within the membrane.
Preliminary Porcine Femoral Segmental Defect Study
A critical-sized porcine segmental defect model was conducted to evaluate the ability of porcine decellularized dermal membrane to aid in the repair of the periosteum from the remaining long bone fragments. The study investigated the ability of the decellularized membrane to protect the bone defect site and minimize fibrous tissue infiltratioiL This preliminary study with two mini-pigs was used to refine the surgical technique for further evaluation of the acellular dermal matrix in the segmental defect model.
The procedure was performed under general inhalation anesthesia using tüetomine HCl/zolazepam HCl (4.4 mg/kg, intramuscularly), xylazine (2.2 mg/kg, intramuscularly), atropine (0.05 mg/kg, intramuscularly), and isoflurane. Veterinary technicians monitored the anesthesia throughout the procedure.
A midshaft, critical-sized, segmental defect measuring in length twotimes the diameter of the bone was surgically created unilaterally in the femur of two male Hanford mini-pigs (8-12 weeks old). A metallic bone plate and screws (Synthes Plate, Synthes, Pa) were applied to fix the osteotomized bone in anatomic position.
The porcine dermal membrane was prepared by soaking it in sterile saline for approximately 10 minutes before surgical application. The membrane was wrapped around the cylindrical bone defect creating a tube. The membrane was overlapped approximately 5 mm on the proximal and distal bone fragments and secured with 4-0 Vicryl sutures in an interrupted pattern circumferenfially, proximally, and distally to the periosteum.
Prior to closure of the membrane tube, the defect defined by the membrane was filled with a 1:1 ratio of OsteoSet Pellets (Wright Medical Technology, Ine) mixed with cancellous autograft bone chips obtained from the proximal humerus. After the defect was filled with the graft materials, the membrane was closed along its length as a seam using 4-0 Vicryl sutures in a continuous pattern.
Following the surgical procedure, analgesia, antibiotics, and intravenous fluids were maintained under the care of veterinary staff. The animals were allowed unrestricted weight bearing and monitored daily by veterinary staff.
Osseous healing of the defect was monitored from clinical radiographs taken immediately postoperative and at 3 and 6 weeks. After 6 weeks, the animals were euthanized using a supersaturated solution of sodium pentobarbital (2.3 cc/10 lb, intravenously) and the femora were removed.
Postmortem high-resolution contact radiographs were obtained of the intact and cross-sectioned isolated femora. The bones were sectioned in the transverse plane for histological analyses to characterize the nature of new bone and the implanted membrane material in the defects. The sections were stained with H&E for study by light microscopy.
Figure 2: Macroimage of defect with porcine dermal membrane covering the porcine femoral drill hole defect.
Histologic results from the athymic rat muscle pouch study indicated a general increase in the infiltration of host cells. The cellular repopulation was higher at the edges of each specimen as compared to the middle due to the access from all the sides of the implant rather than just the top and bottom surfaces.
Upon cross-sectional examination through the thickness of the sheets, higher cellular infiltration was evident on the deep-dermal edge of the material as compared to the basement-membrane edge. Cellular repopulation increased through days 2-21 (Figure I). By day 21, there was a relatively uniform distribution of cells through the thickness of the membranes.
Evidence of revascularization was seen on days 14 and 21 as indicated by the appearance of blood vessels within the matrix. These data indicated that the vasculature was formed between days 7 and 1 4.
Figure 3: Cell repopulation of membrane in the autograft group affixed with Tisseel and tacks at X200 magnification. Figure 4: H&E staining at x200 magnification denotes areas of revascularization within the membrane.
Figure 5: lmmunostaining for factor Vlll-related antigen (brown stain) at ? 200 magnification denotes positive staining vascular enodothelial cells within the membrane.
Figure 6: Radiograph demonstrating defect filled with OsteoSet Pellets and autograft immediately postoperatively and 3 weeks postoperatively. Figure 7: Contact radiographs show an early tubular structure of new bone bridging the defect within the margins of the implanted membrane at 6 weeks.
Figure 8: Cross-sectional contact radiographs demonstrate the struts of new bone formation that are bridging the defect area at 6 weeks.
The preliminary results from the porcine femoral drill hole defect provided evidence of cellular repopulation and revascularization along with the ability to maintain the underlying bone defect site. Gross examination revealed that the membranes were intact and adherent to surrounding bone (Figure 2), and the membranes prevented the underlying bone graft materials from escaping the bone defect site.
Histologic examination revealed intact membranes that were consistently repopulated with viable cells (Figure 3). The blue-stained cells demonstrated a stellate morphology consistent with a fibroblast phenotype. Standard H&E histological stain and factor Vlll-related antigen immunostain indicated evidence of neovasculature within the membrane sheets. The antigen is present within the cytoplasm of endothelial cells. The H&E stain (Figure 4) demonstrated blue-stained nuclei and red-stained red blood cells present within the porcine dermal matrix covering the bone-defect site.
The factor VUI-immunostained scaffold (Figure 5) displayed brownrimmed areas marking the vascular endothelial cells. These data further confirmed the revascularization ability of the dermal scaffold material.
The screening study initiated with the porcine dermal membrane using a critical-sized porcine segmental defect model provided positive indications of bone defect restoration at a 6-week time period. The pigs used in this study resumed weight bearing on the operated limbs within 5 days postoperatively, and the wounds healed in a routine manner.
The representative postoperative and 3-week postoperative clinical radiographs (Figure 6) demonstrated the defects filled with OsteoSet Pellets and autograft. The defects remained stabilized in both pigs without fracture of the bone or breakage of the plates or screws. Each pig had one screw loosen by 3 weeks, and several screws were loosened in one pig at 6 weeks.
After 6 weeks, both pigs had a periosteal reaction over the cranial and lateral aspect of the femur encompassing the plate to varying degrees. Varying amounts of new bone were present in the defects of both pigs. The proximal and distal ends of the native femur exhibited proliferation of bone from the periosteal, cortical, and medullary surfaces. The bone extended into the defect as an initial phase of re-establishing the diaphyseal medullary canal.
Figure 9: The interlaced collagen bundles (A) of the dermal matrix and the newly formed bone are evident (x4 magnification). The membrane appears to be guiding the formation of bone within the margins of the preserved extracellular structures of the collagen matrix. Figure 10: The polarized light image at X4 magnification demonstrates the pattern of interlaced collagen bundles (A) continuing into the bony layer (B). Figure 11: x10 magnification image clearly demonstrates the interwoven structure of the collagen membrane and the newly formed bone. Note the biocompatibility of the membrane evidenced by absence of an intervening layer of fibrous tissue or signs of inflammatory response. Figure 12: Polarized light image at X10 magnification demonstrates the collagen bundles of the matrix continuing into the new bony layer. Figure W. The characteristic pattern o1 the collagen matrix can be seen within this section of newly formed bone at Xl 9 magnification.
Postmortem radiographs (Figure 7) showed a considerable amount of new bone formed in the defect of one pig, resembling an early tubular structure that appeared to penetrate within the margins of the implanted membrane. Although a solid tubular structure was not completely reconstructed at this early 6-week interval, struts of new bone formation were bridging the defect.
The serial cross-sectional radiographs (Figure 8) demonstrated the struts of new bone formation. Radiographically, the other pig appeared to have nonunion at this early time, although small foci of new bone were formed diffusely within the defect.
Histologic data demonstrated thick, interlaced collagen bundles characteristic of the preserved dermal collagen matrix and newly formed bone in the segmental defect (Figure 9). The dermal membrane appeared to guide the new bone formation within the implanted margins.
Polarized light data demonstrated the pattern of interlaced collagen bundles continuing into the bony layer (Figure 10). The interwoven structure of the membrane and the newly formed bone in this segmental defect were clearly demonstrated with highmagnification images (Figures 1 1 and 12). The same observation was noted in another specimen where the characteristic pattern of the extracellular collagen matrix was seen within the newly formed bone (Figure 13).
The data demonstrated new bone formation penetrating within the three-dimensional matrix of the implanted membranes. Within the defects, some of the new bone was formed in the dermal collagen membrane through an initial cartilaginous phase (Figure 14).
The absence of an intervening layer of fibrous tissue was noted in the histologic data as bone was in direct apposition with the dermal membrane. The data also showed the biocompatible nature of the acellular dermal matrix, evidenced by no signs of iirflammatory response.
The athymic rat submuscular implantation of the decellularized human collagen membrane revealed a steady increase in cellular repopulation over the time period tested. From days 2 through 21, the membranes were populated by increasing amounts of host cells over time.
The study indicated that the formation of blood vessels within the preserved channels of the extracellular matrix accrued between days 7 and 14. The porcine femoral drill hole study results also demonstrated that the decellularized membrane repopulated with host cells and revascularized. The data from both of these studies confirmed the ability of the acellular dermal scaffold to rapidly repopulate with cells and revascularize.
Researchers have highlighted the importance of autologous periosteum to provide vascularity and nutrional diffusion to underlying bone.1,2 A material used to augment damaged or inadequate periosteum when autologous periosteum is not available should rapidly repopulate with cells and revascularize.
The processing technology used in the production of the acellular dermal scaffold preserves the biochemical and extracellular structural properties of the dermal collagen matrix (Figure 15). The data from these studies confirmed that the intact extracellular matrix provided a template for revascularization that may lead to subsequent nutrional diffusion to the underlying bone site.
Results from the porcine femoral drill hole study and segmental study indicated that the decellularized dermal membrane provided a protected environment for healing as the underlying graft materials were maintained at the defect site. The maintenance of the underlying bone site using the dermal matrix supported the concept that the membrane may possess important attributes needed for periosteum replacement in situations of damaged or inadequate autologous periosteum.
Grundnes and Reikeras3 discussed the importance of obtaining a periosteal seal that prevents local hematoma and mesenchymal cells from escaping into the local soft tissue. Their concept may be associated with maintenance of the bone site.
Figure 14: Some of the newly formed bone within the collagen membrane appears to form through an initial cartilaginous phase (x 38 magnification).
Figure 15: Scanning electron microscopy image ?400 magnification illustrates a crosssectional view of the intact tissue matrix (A) and a surface view of the matrix highlights a preserved vascular channel (B).
Eyre-Brook1 examined the importance of autologous periosteum serving as a connection between fragments of bone and delineating a cavity to assist remaining callus edges to bridge. It is inferred that a replacement material to augment damaged or inadequate periosteum may need to possess attributes that facilitate guided bone regeneration.
The preliminary porcine segmental defect study provided positive indications of the ability of the deceífuíarized matrix with preserved extracellular structure to allow for bone defect restoration in a challenging segmental model.
The radiographic and histologic data indicated that the graft material remained at the defect site, and the early phase of bone defect healing was apparent. There was abundant cellular activity noted within the dermal membrane. In fact, new bone formation penetrated within the implanted membrane. There appeared to be active incorporation of the collagen matrix into the new bone, and new bone was found to form within the margins of implanted tubular membrane structure.
The histological sections provided a preliminary indication that the decellularized dermal matrix may function as a biochemical and physical guide for new bone formation. A previous study using the dermal matrix for guided bone regeneration in the staged placement of a dental implant indicated the material allowed for bone regeneration and uneventful healing.9
Previous studies by Macnab and De Haas2 showed the importance of obtaining a periosteal seal with autologous periosteum to minimize infiltration of fibrous tissue invasion between bone ends. A material used to augment damaged or inadequate perìostuem, when autologous periosteum is not available, should minimize the infiltration of fibrous tissue.
The data from the porcine segmental defect study revealed a lack of fibrous tissue infiltration in the bone defect site as evidenced by new bone adjacent to the implanted dermal membrane.
The preclinical data presented for the acellular dermal matrix show that the material possesses many important attributes to function as a scaffold for periosteum replacement. The acellular dermal scaffold allowed for repopulation of host cells, revascularization, protection of the bone environment for healing and restoration, and minimization of fibrous tissue invasion.
The maintenance of autologous periosteum in orthopedic surgical procedures may not always be possible. Therefore, a material used to augment or replace damaged or inadequate periosteum should possess as many of the attributes of autologous periosteum as possible. The Graftjacket Acellular Periosteum Replacement Scaffold possesses many of the important attributes needed for periosteum replacement. Further studies to assess the role of acellular dermal matrix in a porcine segmental defect model are underway.
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