Karl H. Kraus

The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects

Bone marrow has been shown to contain a population of rare mesenchymal stem cells that are capable of forming bone, cartilage, and other connective tissues. We examined the effect of cultured autologous mesenchymal stem cells on the healing of critical-sized (twenty-one-millimeter-long) segmental defects in the femora of adult female dogs. Autologous mesenchymal stem cells were isolated from bone marrow, grown in culture, and loaded onto porous ceramic cylinders consisting of hydroxyapatite (65 per cent) and &bgr;-tricalcium phosphate ceramic (35 per cent). The animals were randomly assigned to one of three groups. In Group A (six dogs), a porous ceramic cylinder that had been loaded with autologous mesenchymal stem cells was implanted in the defect. In Group B (six dogs), a ceramic cylinder that had not been loaded with cells was placed in the defect. In Group C (three dogs), the defect was left untreated (no ceramic cylinder was implanted). Radiographs were made immediately after the operation and at four-week intervals. At sixteen weeks, the animals were killed, the involved femora were removed, and undecalcified histological sections from the defects and adjacent bone were prepared. Histological and histomorphometric studies were carried out to examine the healing of the defects and the formation of bone in and around the ceramic implants. Atrophic non-union occurred in all of the femora that had untreated defects, and only a small amount of trabecular bone formed at the cut ends of the cortex of the host bone in this group. In contrast, radiographic union was established rapidly at the interface between the host bone and the implants that had been loaded with mesenchymal stem cells. Numerous fractures, which became more pronounced with time, developed in the implants that had not been loaded with cells. Histological and morphometric analyses demonstrated that both woven and lamellar bone had filled the pores of the implants that had been loaded with mesenchymal stem cells; the amount of bone was significantly greater (p < 0.05) than that found in the pores of the implants that had not been loaded with cells. In addition, a large collar of bone (mean maximum thickness, 3.14 millimeters) formed around the implants that had been loaded with cells; this collar became integrated and contiguous with callus that formed in the region of the periosteum of the host bone. The collar of bone remodeled during the sixteen-week period of study, resulting in a size and shape that were comparable with those of the segment of bone that had been resected. Callus did not develop around the cortex of the host bone or around the defect in any of the specimens in the other two groups. CLINICAL RELEVANCE: Autologous cultured bone-marrow-derived mesenchymal stem cells that had been loaded onto porous ceramic cylinders elicited the healing of critical-sized segmental bone defects in dogs. It may be possible to exploit this technology to elicit the healing of bone defects in humans by using cells from bone marrow that has been aspirated from the iliac crest of the patient. This approach may provide an alternative to autologous bone-grafting and may be particularly useful when the number of endogenous mesenchymal stem cells is relatively small.

Allogeneic Mesenchymal Stem Cells Regenerate Bone in a Critical-Sized Canine Segmental Defect

BACKGROUND Mesenchymal stem cells from adult bone marrow are multipotent cells capable of forming bone, cartilage, and other connective tissues. In a previous study, we demonstrated that autologous mesenchymal stem cells could repair a critical-sized bone defect in the dog. The objective of this study was to determine whether the use of allogeneic mesenchymal stem cells could heal a critical-sized bone defect in the femoral diaphysis in dogs without the use of immunosuppressive therapy. METHODS A critical-sized segmental bone defect, 21 mm in length, was created in the mid-portion of the femoral diaphysis of twelve adult dogs that weighed between 22 and 25 kg. Each defect was treated with allogeneic mesenchymal stem cells loaded onto a hollow ceramic cylinder consisting of hydroxyapatite-tricalcium phosphate. A complete mismatch between donor stem cells and recipient dogs was identified by dog leukocyte antigen typing prior to implantation. The healing response was evaluated histologically and radiographically at four, eight, and sixteen weeks after implantation. The radiographic and histological results at sixteen weeks were compared with the historical data for the control defects, which included defects that had been treated with a cylinder loaded with autologous mesenchymal stem cells, defects treated with a cylinder without mesenchymal stem cells, and defects that had been left untreated (empty). The systemic immune response was evaluated by the analysis of recipient serum for production of antibodies against allogeneic cells. RESULTS For defects treated with allogeneic mesenchymal stem cell implants, no adverse host response could be detected at any time-point. Histologically, no lymphocytic infiltration occurred and no antibodies against allogeneic cells were detected. Histologically, by eight weeks, a callus spanned the length of the defect, and lamellar bone filled the pores of the implant at the host bone-implant interface. Fluorescently labeled allogeneic cells were also detected. At sixteen weeks, new bone had formed throughout the implant. These results were consistent with those seen in implants loaded with autologous cells. Implants loaded with allogeneic or autologous stem cells had significantly greater amounts of bone within the available pore space than did cell-free implants at sixteen weeks (p < 0.05). CONCLUSIONS The results of this study demonstrated that allogeneic mesenchymal stem cells loaded on hydroxyapatite-tricalcium phosphate implants enhanced the repair of a critical-sized segmental defect in the canine femur without the use of immunosuppressive therapy. No adverse immune response was detected in this model.

Mesenchymal stem cells in osteobiology and applied bone regeneration

Bone marrow contains a population of rare progenitor cells capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. These cells, referred to as MSCs, can be purified and culture expanded from animals and humans. This review summarizes recent experimentation focused on characterizing the cellular aspects of osteogenic differentiation, and exploration of the potential for using autologous stem cell therapy to augment bone repair and regeneration. The authors have completed an array of preclinical studies showing the feasibility and efficacy of MSC based implants to heal large osseous defects. After confirming that syngeneic rat MSCs could heal a critical size segmental defect in the femur, it was established that human MSCs form bone of considerable mechanical integrity when implanted in an osseous defect in an immunocompromised animal. Furthermore, bone repair studies in dogs verify that the technology is transferable to large animals, and that the application of this technology to patients at geographically remote sites is feasible. These studies suggest that by combining MSCs with an appropriate delivery vehicle, it may be possible to offer patients new therapeutic options.

Gel Matrix Vehicles for Growth Factor Application in Nerve Gap Injuries Repaired with Tubes: A Comparison of Biomatrix, Collagen, and Methylcellulose

The repair of nerve gap injuries with tubular nerve guides has been used extensively as an in vivo test model in identifying substances which may enhance nerve regeneration. The model has also been used clinical nerve repair. The objective of this study was to compare three different gel matrix-forming materials as potential vehicles for growth factors in this system. The vehicles included a laminin containing extracellular matrix preparation (Biomatrix), collagen, and a 2% methylcellulose gel. The growth factor test substance consisted of a combination of platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor I (IGF-I). An 8-mm gap in rat sciatic nerve was repaired with a silicone tube containing each of the vehicles alone or with a combination of each vehicle plus PDGF-BB and IGF-I. At 4 weeks after injury, the application of the growth factor combination significantly stimulated axonal regeneration when applied in methylcellulose or collagen, but not in Biomatrix. A similar trend was present between the vehicle control groups. By 8 weeks after injury, nerves repaired with methylcellulose as a vehicle had significantly greater conduction velocity than either collagen or Biomatrix. It was concluded that a 2% methylcellulose gel was the best of the three matrices tested, both in its effects on nerve regeneration and flexibility of formulation.

Fluorescently labeled mesenchymal stem cells (MSCs) maintain multilineage potential and can be detected following implantation into articular cartilage defects

Several studies have reported enhanced repair of damaged cartilage following implantation of mesenchymal stem cells (MSCs) into full-thickness cartilage defects suggesting that the cells in the repair tissue were derived from the implant. However, it cannot be excluded that the enhanced tissue repair is derived from host cells recruited to the defect in response to the implant, rather than the re-population of the tissue by the implanted MSCs. Our objective was to study the short-term fate of fluorescently labeled MSCs after implantation into full-thickness cartilage defects in vivo. The fluorescent dye used in our studies did not affect MSC viability or their ability to undergo osteogenic and chondrogenic differentiation in vitro. MSC gelatin constructs were implanted into full-thickness cartilage defects in goats. These cells retained the dye and were detectable by histology and flow cytometry. At intervals spanning 2 weeks post-implantation we observed gradual loss of implanted cells in the defect as well as fragments of gelatin sponge containing labeled MSCs in deep marrow spaces indicating fragmentation, dislodgement and passive migration. Fluorescent labeling enabled us to determine whether the implanted cells were lost during early time points after implantation as well as their spatial orientation throughout the defect. By determining the fate of implanted cells, new biomaterials could be engineered to correct undesirable characteristics. Testing of new biomaterials in short-term in vivo models would provide faster optimization for cell retention needed for successful, long-term cartilage regeneration.

The use of a cap-shaped coil for transcranial magnetic stimulation of the motor cortex.

A cap-shaped coil is introduced as a superior design for inducing transcranial magnetic motor evoked potentials for spinal cord monitoring. Evaluation of the magnetic characteristics of the cap coil showed higher induced electrical fields at and below the depth of the cortical surface, compared to a 9-cm, butterfly-shaped coil. Twenty normal adults were stimulated with the cap coil and a 9-cm round coil in three positions. Compound muscle action potentials were recorded from the left and right abductor digiti minimi and anterior tibialis muscles. The cap coil induced potentials with higher intensities and lower variability between consecutive stimuli. The cap coil was also more able to simultaneously induce motor evoked potentials from the four muscles studied. This coil design should provide superior means of inducing transcranial magnetic motor evoked potentials in multiple muscles.

The pathophysiology of spinal cord injury and its clinical implications.

The pathophysiology of spinal cord injury can be categorized as acute impact or compression. Acute impact injury is a concussion of the spinal cord. This type of injury initiates a cascade of events focused in the gray matter, and results in hemorrhagic necrosis. The initiating event is a hypoperfusion of the gray matter. Increases in intracellular calcium and reperfusion injury play key roles in cellular injury, and occur early after injury. The extent of necrosis is contingent on the amount of initial force of trauma, but also involves concomitant compression, perfusion pressures and blood flow, and administration of pharmacological agents. Preventing or quelling this cascade of events must involve mechanisms occurring in the initial stages. Spinal cord compression occurs when a mass impinges on the spinal cord causing increased parenchymal pressure. The tissue response is gliosis, demyelination, and axonal loss. This occurs in the white matter, whereas gray matter structures are preserved. Rapid or a critical degree of compression will result in collapse of the venous side of the microvasculature, resulting in vasogenic edema. Vasogenic edema exacerbates parenchymal pressure, and may lead to rapid progression of disfunction. Treatment of compression should focus on removal of the offending mass.

Critically sized osteo-periosteal femoral defects: a dog model.

A 21-mm defect was created in 1 femoral diaphysis each of 15 dogs. Periosteum as well as a cylinder of bone was removed, and the defect was stabilized with a bone plate. Twelve of the defects were filled with an equal volume of autogenous cancellous bone harvested from the ipsilateral ilium. Three defects were left untreated. Cranial to caudal radiographs were taken postoperatively and every 4 weeks for 16 weeks. The radiographs were evaluated for healing using two ordinal scales. At 16 weeks, the dogs were euthanized and the femurs harvested for biomechanical testing and histologic evaluation. Both operated and contralateral not operated femurs were mechanically tested to failure in torsion, and load at failure and stiffness were calculated. All dogs tolerated the procedure well, and were using the operated limb within 1 or 2 days postoperatively. There were no complications noted during the 16 weeks of the study. Unfilled defects did not heal and became atrophic nonunions. The defects filled with autogenous cancellous bone healed in a consistent pattern of consolidation, incorporation, and remodeling, with uniform increases of both ordinal scales used. The femoral cortex opposite the bone plate demonstrated most mature remodeling, evident both radiographically as well as histologically. Unoperated femurs failed at 13.61 +/- 3.88 N-m and grafted femurs failed at 2.96 +/- 1.3 N-m, which was 23% of the measurement of the unoperated femur. Relative stiffness of the unoperated femurs was 5974 +/- 4316 N-m2/radian, and grafted femurs had a relative stiffness of 642 +/- 561 N-m2/radian, which was 10.4% of the measurement of unoperated femur. This model proved to be a critically sized defect, which when left unfilled resulted in an atrophic nonunion, and when filled with cancellous bone resulted in a consistent healing pattern.

Factors associated with recovery from paraplegia in dogs with loss of pain perception in the pelvic limbs following intervertebral disk herniation

OBJECTIVE To investigate associations between recovery of locomotion and putative prognostic factors in dogs with loss of deep pain perception in the pelvic limbs caused by intervertebral disk herniation (IVDH). DESIGN Prospective cohort study. ANIMALS 78 client-owned dogs evaluated for IVDH that underwent spinal decompression surgery. PROCEDURES Dogs with complete loss of deep pain perception in the pelvic limbs and tail underwent routine examinations, advanced imaging, and spinal decompression surgery in accordance with standards of practice and owner consent. For each dog, information was prospectively collected on duration of clinical signs prior to onset of paraplegia; delay between onset of paraplegia and initial referral evaluation; date of recovery of locomotion, death, or euthanasia (3-month follow-up period); and whether dogs had received corticosteroid drugs before surgery. Severity of spinal cord compression at the lesion epicenter was measured via CT or MRI. RESULTS 45 of 78 (58%) of dogs recovered the ability to ambulate independently within 3 months after spinal decompression surgery. No evidence of prognostic value was identified for any of the investigated factors; importantly, a greater delay between onset of paraplegia and referral evaluation was not associated with a poorer prognosis. CONCLUSIONS AND CLINICAL RELEVANCE In this group of dogs with IVDH, immediacy of surgical treatment had no apparent association with outcome. The prognosis for recovery may instead be strongly influenced by the precise nature of the initiating injury.

Peripheral ischemia as a complicating factor during somatosensory and motor evoked potential monitoring of aortic surgery

HE incidence of paraplegia following aortic surgery is reported to vary between 2% and 25%.lm4 This reported incidence has made intraoperative spinal cord monitoring an area of interest.5-9 Two monitoring modalities, somatosensory evoked potentials (SEP) and motor evoked potentials (MEP), can detect ischemia of nervous system structures. Of these, the SEP, although controversial, is used most for both experimental and clinical monitoring of aortic aneurysm surgery.5-i0 The SEP monitors physiologic integrity of the peripheral nerve, from the site of stimulation to the cerebral cortex, via the dorsal columns of the spinal cord. The most sensitive area of the spinal cord to ischemia is the anterior horn area, as shown by the distribution of postoperative spinal cord injury.” Although some investigators have suggested that a steal of blood from the posterior to anterior spinal cord may occur during aortic cross-clamping, changes in SEPs will, at best, indirectly monitor ischemia of the motor areas1*J3 Hence, SEP monitoring has yielded low specificity and sensitivity to postoperative neurologic deficits in the absence of shunt or partial bypass use during these operations.r4J5 As suggested by McNulty et a1,r3 use of these operative adjuncts, however, does in part improve the predictive value of SEP monitoring for postoperative neurologic deficits. Recent investigations have used MEPs induced by electrical stimulation to monitor spinal cord function during aortic occlusion.16-ia Monitoring MEPs may be more sensitive than SEPs to spinal cord ischemia. Clinical examples using magnetic transcranially induced MEPs in humans during aortic aneurysm surgery have yet to be reported. An often neglected factor in spinal cord monitoring is that an insult to any part of the nervous system pathway that is monitored will result in a change in the evoked potentials.19 Ischemia to the peripheral nervous system is an important factor that must be addressed during future clinical investigations involving evoked potential monitoring of aortic aneurysm surgery. Two cases are presented, one in which SEPs alone and one in which both SEPs and MEPs were monitored during aortic surgery. Both cases demonstrate the importance of peripheral ischemia as a possible explanation for changes in SEPs and/or MEPs during aortic surgery.