Recent Progress in Animal Studies of the Skin- and Bone-integrated Pylon With Deep Porosity for Bone-Anchored Limb Prosthetics With and Without Neural Interface.

INTRODUCTION: The three major unresolved problems in bone-anchored limb prosthetics are stable, infection-free integration of skin with a percutaneous bone implant, robust skeletal fixation between the implant and host bone, and a secure interface of sensory nerves and muscles with a prosthesis for the intuitive bidirectional prosthetic control. Here we review results of our completed work and report on recent progress. MATERIALS AND METHODS: Eight female adult cats received skin- and bone-integrated pylon (SBIP) and eight male adult cats received SBIP-peripheral neural interface (PNI) pylon into the right distal tibia. The latter pylons provided PNI for connection between a powered sensing transtibial prosthesis and electrodes in residual soleus muscle and on residual distal tibial nerve. If signs of infection were absent 28-70 days after implantation, cats started wearing a passive prosthesis. We recorded and analyzed full-body mechanics of level and slope locomotion in five cats with passive prostheses and in one cat with a powered sensing prosthesis. We also performed histological analyses of tissue integration with the implants in nine cats.Four pigs received SBIPs into the left hindlimb and two pigs-into the left forelimb. We recorded vertical ground reaction forces before amputation and following osseointegration. We also conducted pullout postmortem tests on the implanted pylons. One pig received in dorsum the modified SBIPs with and without silver coating. RESULTS: Six cats from the SBIP groups had implant for 70 days. One cat developed infection and did not receive prosthesis. Five cats had pylon for 148 to 183 days, showed substantial loading of the prosthesis during locomotion (40.4% below presurgery control), and demonstrated deep ingrowth of skin and bone tissue into SBIP (over 60%). Seven of eight cats from the SBIP-PNI group demonstrated poor pylon integration without clinical signs of infection. One cat had prosthesis for 824 days (27 months). The use of the bidirectionally controlled prosthesis by this animal during level walking demonstrated increased vertical loading to nearly normal values, although the propulsion force was significantly reduced.From the study on pigs, it was found that symmetry in loading between the intact and prosthetic limbs during locomotion was 80 ± 5.5%. Skin-implant interface was infection-free, but developed a stoma, probably because of the high mobility of the skin and soft tissues in the pig's thigh. Dorsal implantation resulted in the infection-free deep ingrowth of skin into the SBIP implants. CONCLUSIONS: Cats with SBIP (n = 5) and SBIP-PNI (n = 1) pylons developed a sound interface with the residuum skin and bone and demonstrated substantial loading of prosthetic limb during locomotion. One animal with SBIP developed infection and seven cats with SBIP-PNI demonstrated poor bone integration without signs of infection. Future studies of the SBIP-PNI should focus on reliability of integration with the residuum. Ongoing study with pigs requires decreasing the extra mobility of skin and soft tissues until the skin seal is developed within the SBIP implant.

Kinetics of individual limbs during level and slope walking with a unilateral transtibial bone-anchored prosthesis in the cat.

Ongoing animal preclinical studies on transcutaneous bone-anchored prostheses have aimed to improve biomechanics of prosthetic locomotion in people with limb loss. It is much less common to translate successful developments in human biomechanics and prosthetic research to veterinary medicine to treat animals with limb loss. Current standard of care in veterinary medicine is amputation of the whole limb if a distal segment cannot be salvaged. Bone-anchored transcutaneous prostheses, developed for people with limb loss, could be beneficial for veterinary practice. The aim of this study was to examined if and how cats utilize the limb with a bone-anchored passive transtibial prosthesis during level and slope walking. Four cats were implanted with a porous titanium implant into the right distal tibia. Ground reaction forces and full-body kinematics were recorded during level and slope (±50%) walking before and 4-6 months after implantation and prosthesis attachment. The duty factor of the prosthetic limb exceeded zero in all cats and slope conditions (p < 0.05) and was in the range of 45.0-60.6%. Thus, cats utilized the prosthetic leg for locomotion instead of walking on three legs. Ground reaction forces, power and work of the prosthetic limb were reduced compared to intact locomotion, whereas those of the contralateral hind- and forelimbs increased (p < 0.05). This asymmetry was likely caused by insufficient energy generation for propulsion by the prosthetic leg, as no signs of pain or discomfort were observed in the animals. We concluded that cats could utilize a unilateral bone-anchored transtibial prosthesis for quadrupedal level and slope locomotion.

Accurate stepping on a narrow path: mechanics, EMG, and motor cortex activity in the cat.

How do cats manage to walk so graciously on top of narrow fences or windowsills high above the ground while apparently exerting little effort? In this study we investigated cat full-body mechanics and the activity of limb muscles and motor cortex during walking along a narrow 5-cm path on the ground. We tested the hypotheses that during narrow walking 1) lateral stability would be lower because of the decreased base-of-support area and 2) the motor cortex activity would increase stride-related modulation because of imposed demands on lateral stability and paw placement accuracy. We measured medio-lateral and rostro-caudal dynamic stability derived from the extrapolated center of mass position with respect to the boundaries of the support area. We found that cats were statically stable in the frontal plane during both unconstrained and narrow-path walking. During narrow-path walking, cats walked slightly slower with more adducted limbs, produced smaller lateral forces by hindlimbs, and had elevated muscle activities. Of 174 neurons recorded in cortical layer V, 87% of forelimb-related neurons (from 114) and 90% of hindlimb-related neurons (from 60) had activities during narrow-path walking distinct from unconstrained walking: more often they had a higher mean discharge rate, lower depth of stride-related modulation, and/or longer period of activation during the stride. These activity changes appeared to contribute to control of accurate paw placement in the medio-lateral direction, the width of the stride, rather than to lateral stability control, as the stability demands on narrow-path and unconstrained walking were similar.

Body stability and muscle and motor cortex activity during walking with wide stance.

Biomechanical and neural mechanisms of balance control during walking are still poorly understood. In this study, we examined the body dynamic stability, activity of limb muscles, and activity of motor cortex neurons [primarily pyramidal tract neurons (PTNs)] in the cat during unconstrained walking and walking with a wide base of support (wide-stance walking). By recording three-dimensional full-body kinematics we found for the first time that during unconstrained walking the cat is dynamically unstable in the forward direction during stride phases when only two diagonal limbs support the body. In contrast to standing, an increased lateral between-paw distance during walking dramatically decreased the cat's body dynamic stability in double-support phases and prompted the cat to spend more time in three-legged support phases. Muscles contributing to abduction-adduction actions had higher activity during stance, while flexor muscles had higher activity during swing of wide-stance walking. The overwhelming majority of neurons in layer V of the motor cortex, 82% and 83% in the forelimb and hindlimb representation areas, respectively, were active differently during wide-stance walking compared with unconstrained condition, most often by having a different depth of stride-related frequency modulation along with a different mean discharge rate and/or preferred activity phase. Upon transition from unconstrained to wide-stance walking, proximal limb-related neuronal groups subtly but statistically significantly shifted their activity toward the swing phase, the stride phase where most of body instability occurs during this task. The data suggest that the motor cortex participates in maintenance of body dynamic stability during locomotion.

An animal model to evaluate skin-implant-bone integration and gait with a prosthesis directly attached to the residual limb.

BACKGROUND: Despite the number of advantages of bone-anchored prostheses, their use in patients is limited due to the lack of complete skin-implant integration. The objective of the present study was to develop an animal model that would permit both detailed investigations of gait with a bone-anchored limb prosthesis and histological analysis of the skin-implant-bone interface after physiological loading of the implant during standing and walking. METHODS: Full-body mechanics of walking in two cats were recorded and analyzed before and after implantation of a percutaneous porous titanium pylon into the right tibia and attachment of a prosthesis. The rehabilitation procedures included initial limb casting, progressively increasing loading on the implant, and standing and locomotor training. Detailed histological analysis of bone and skin ingrowth into implant was performed at the end of the study. FINDINGS: The two animals adopted the bone-anchored prosthesis for standing and locomotion, although loads on the prosthetic limb during walking decreased by 22% and 62%, respectively, 4months after implantation. The animals shifted body weight to the contralateral side and increased propulsion forces by the contralateral hindlimb. Histological analysis of the limb implants demonstrated bone and skin ingrowth. INTERPRETATION: The developed animal model to study prosthetic gait and tissue integration with the implant demonstrated that porous titanium implants may permit bone and skin integration and prosthetic gait with a bone-anchored prosthesis. Future studies with this model will help optimize the implant and prosthesis properties.

Effects of pore size, implantation time, and nano-surface properties on rat skin ingrowth into percutaneous porous titanium implants.

The main problem of percutaneous osseointegrated implants is poor skin-implant integration, which may cause infection. This study investigated the effects of pore size (Small, 40-100 µm and Large, 100-160 µm), nanotubular surface treatment (Nano), and duration of implantation (3 and 6 weeks) on skin ingrowth into porous titanium. Each implant type was percutaneously inserted in the back of 35 rats randomly assigned to seven groups. Implant extrusion rate was measured weekly and skin ingrowth into implants was determined histologically after harvesting implants. It was found that all three types of implants demonstrated skin tissue ingrowth of over 30% (at week 3) and 50% (at weeks 4-6) of total implant porous area under the skin; longer implantation resulted in greater skin ingrowth (p < 0.05). Only one case of infection was observed (infection rate 2.9%). Small and Nano groups showed the same implant extrusion rate which was lower than the Large group rate (0.06 ± 0.01 vs. 0.16 ± 0.02 cm/week; p < 0.05). Ingrowth area was comparable in the Small, Large, and Nano implants. However, qualitatively, the Nano implants showed greatest cellular inhabitation within first 3 weeks. We concluded that percutaneous porous titanium implants allow for skin integration with the potential for a safe seal.

Locomotor changes in length and EMG activity of feline medial gastrocnemius muscle following paralysis of two synergists.

The mechanism of the compensatory increase in electromyographic activity (EMG) of a cat ankle extensor during walking shortly after paralysis of its synergists is not fully understood. It is possible that due to greater ankle flexion in stance in this situation, muscle spindles are stretched to a greater extent and, thus, contribute to the EMG enhancement. However, also changes in force feedback and central drive may play a role. The aim of the present study was to investigate the short-term (1- to 2-week post-op) effects of lateral gastrocnemius (LG) and soleus (SO) denervation on muscle fascicle and muscle-tendon unit (MTU) length changes, as well as EMG activity of the intact medial gastrocnemius (MG) muscle in stance during overground walking on level (0%), downslope (-50%, presumably enhancing stretch of ankle extensors in stance) and upslope (+50%, enhancing load on ankle extensors) surfaces. Fascicle length was measured directly using sonomicrometry, and MTU length was calculated from joint kinematics. For each slope condition, LG-SO denervation resulted in an increase in MTU stretch and peak stretch velocity of the intact MG in early stance. MG muscle fascicle stretch and peak stretch velocity were also higher than before denervation in downslope walking. Denervation significantly decreased the magnitude of MG fascicle shortening and peak shortening velocity during early stance in level and upslope walking. MG EMG magnitude in the swing and stance phases was substantially greater after denervation, with a relatively greater increase during stance of level and upslope walking. These results suggest that the fascicle length patterns of MG muscle are significantly altered when two of its synergists are in a state of paralysis. Further, the compensatory increase in MG EMG is likely mediated by enhanced MG length feedback during downslope walking, enhanced feedback from load-sensitive receptors during upslope walking and enhanced central drive in all walking conditions.

Distinct muscle fascicle length changes in feline medial gastrocnemius and soleus muscles during slope walking.

On the basis of differences in physiology, e.g., histochemical properties and spindle density, and the structural design of the cat soleus (SO) and medial gastrocnemius (MG) muscles, we hypothesized that 1) fascicle length changes during overground walking would be both muscle and slope dependent, which would have implications for the muscles' force output as well as sensory function, and that 2) muscle-tendon unit (MTU) and fascicle length changes would be different, in which case MTU length could not be used as an indicator of muscle spindle strain. To test these hypotheses, we quantified muscle fascicle length changes and compared them with length changes of the whole MTU in the SO and MG during overground walking at various slopes (0, +/- 25, +/- 50, +75, and +100%). The SO and MG were surgically instrumented with sonomicrometry crystals and fine-wire electromyogram electrodes to measure changes in muscle fascicle length and muscle activity, respectively. MTU lengths were calculated using recorded ankle and knee joint angles and a geometric model of the hindlimb. The resultant joint moments were calculated using inverse dynamics analysis to infer muscle loading. It was found that although MTU length and velocity profiles of the SO and MG appeared similar, length changes and velocities of muscle fascicles were substantially different between the two muscles. Fascicle length changes of both SO and MG were significantly affected by slope intensity acting eccentrically in downslope walking (-25 to -50%) and concentrically in upslope walking (+25 to +100%). The differences in MTU and fascicle behaviors in both the SO and MG muscles during slope walking were explained by the three distinct features of these muscles: 1) the number of joints spanned, 2) the pennation angle, and 3) the in-series elastic component. It was further suggested that the potential role of length feedback from muscle spindles is both task and muscle dependent.