Watch Dr. Sarah Greising discuss the current pathophysiologic understanding of the skeletal muscle remaining following traumatic musculoskeletal injuries. Volumetric muscle loss (VML) injuries result in the abrupt loss of skeletal muscle fibers, causing chronic functional disability in part due to limited muscle regeneration and vast co-morbidities. With a focus on clinically relevant outcome measurements for skeletal muscle function in both small and large animal models of VML injury, this webinar presents various near-term interventions for the restoration of tissue function following complex injuries. Interventions evaluated focus on regenerative rehabilitation approaches using regenerative pharmaceuticals to correct underlying muscle pathophysiology.

1. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Functional Recovery of the
Musculoskeletal System
Following Injury – Leveraging
the Large Animal Model
Sarah Greising, PhD
Kinesiology
University of Minnesota
Associate Professor

2. Functional Recovery of the
Musculoskeletal System Following Injury -
Leveraging the Large Animal Model
Sarah Greising, PhD
Associate Professor
Henry L. Taylor-Arthur S. Leon Professorship
grei0064@umn.edu
08. December. 2022

3. Skeletal muscle function
and physiology Limited regenerative
potential of skeletal
muscle
VML injury
Small to large animal models
Functional recovery

4. Grogan & Hsu, J Am Acad Orthop Surg 2011

5. Injury in which a critical portion of a muscle or muscle
unit is abruptly removed due to trauma or surgery
Outcomes associated with VML injury
• Persistent functional deficits
• Loss of range of motion
• Chronic disability
Gentile et al., 2014 Garg et al., J Ortho Res 2015
Irrecoverable loss of
contractile tissue and
strength
No current clinical, surgical, or
rehabilitative standard of care
that address the loss of
endogenous material
necessary for muscle
regeneration or directly impact
the soft tissue

6. Loss of Strength
Shift of Angle of
Peak Torque
“Longer Muscle Length”
Loss of Active
Range of Motion
Garg et al., J Ortho Res 2015
Biodex Isokinetic testing
60˚/s; 10 Work Loops;
Start at 0˚ - End active range of plantarflexion

7. • Military trauma
On an annual basis VML can result secondary to:
• 150,000 open fractures
• 36,000 industrial or farm accidents
• 30,000 gunshot wounds and related trauma
• 80 shark attacks
• 13,000 soft-tissue cancers
• 4,000 cleft lip and palate
Trauma
Physiologic

8. Orthopaedic Trauma
US economic burden to trauma is in excess of $400 billion yearly
Soft tissue trauma
• 2 million hospitalizations
• 9 million bed days
• 6.5 million outpatient visits
• 18 million emergency room visits
• 64 million physician's office visits
US Department of Defense
• $42.2 billion in initial care
• $108.8 billion in lifetime disability benefit cost to injured service
members

9. The outcomes of these various forms of orthopaedic injuries is chronic
disability in which muscle regeneration is not possible
1) Surgical, regenerative medicine, and/or rehabilitation approach targeted at
regenerating the lost muscle
2) Approaches to improve the remaining muscle and prevent further
degradation, i.e., remodel and/or adapt to the new cellular condition
3) Understand the pathophysiology of the both the injury and muscle
remaining
4) How to improve function, i.e., functional recovery, chronically after injury

10. Various models in literature
• Abdominal wall, latissimus dorsi,
tibialsis anterior, quadriceps,
posterior compartment muscles
• 10-50% of the muscle volume
with functional deficits exceeding
the volume lost
Southern et al., Sci Rep 2019
Small & Large Animal Model of VML Injury

11. Small & Large Animal Model of VML Injury
Porcine peroneus
tertius muscle (~5
g; 20% volume)
Mouse & rat tibialis anterior (TA) muscle Mouse gastrocnemius,
soleus, & plantaris muscles
Pollet & Corona, MMB 2016 & Greising et al., Sci Rep 2017

12. Chronic functional
impairment
Long-term dysfunction, reduced mobility
and physical activity, co-morbidities,
delayed amputation
Chronic loss of
skeletal muscle
Pathophysiology
Clinical Distinction
Irrecoverable
Volumetric
muscle loss
injury
Lack of endogenous
regenerative ability
Increased
pathologic fibrosis
Chronic and heightened
inflammation
Altered muscle
architecture/force transmission
Chronic neural
dysfunction
Metabolic maladaptivity
Satellite cell dysfunction
Clinical Outcomes
Inability to respond to
rehabilitation

13. Sorensen et al., 2022

14. Corona et al., JoVE 2021

15. Pollet & Corona, MMB 2016
Corona et al., JoVE 2021

16. Corona et al., JoVE 2021
Length control

17. Corona et al., JoVE 2021

18. Chronic functional
impairment
Long-term dysfunction, reduced mobility
and physical activity, co-morbidities,
delayed amputation
Chronic loss of
skeletal muscle
Pathophysiology
Clinical Distinction
Irrecoverable
Volumetric
muscle loss
injury
Lack of endogenous
regenerative ability
Increased
pathologic fibrosis
Chronic and heightened
inflammation
Altered muscle
architecture/force transmission
Chronic neural
dysfunction
Metabolic maladaptivity
Satellite cell dysfunction
Clinical Outcomes
Inability to respond to
rehabilitation

19. 1) Simple example of scale up with a HGF loaded biomaterial
2) Complex approach of scale up with pharmacologic treatment
of fibrosis
3) Pathophysiologic understand of impairments to support
future scale up
a) Neural
b) Metabolic
4) Summary and next steps

20. Combined treatment
approach

21. Increased pathologic
fibrosis
An overaccumulation of
extracellular matrix
deposition during the
repair process
Impaired healing and
regeneration

22. Greising et al., BMC Musc 2018
Mouse VML model
 2 Fold increase in
passive torque
 1-2 Fold increase in
collagen content
 Indicating increased
stiffness

23. Hoffman et al., Con Tiss Res 2021
 Increased
fibrosis and
denser collagen
in the muscle
remaining
 Suggesting
increased
stiffness

24.  Conserved increase in collagen
content between the rodent and
pig
 Suggesting increased stiffness
Corona et al., Tis Eng A 2020

25. Awasthi et al., Onco Targets Therp 2015
Anti-Fibrotic Treatment Nintedanib
Intracellular tyrosine kinase inhibitor that
targets fibroblast growth factor receptor
(FGFR) 1-4, platelet-derived growth
factor receptor (PDGFR) α/β, vascular
endothelial growth factor receptor
(VEGFR) 1-3, and the Src family of
tyrosine kinases Lck, Lyn, and Flt-3
I
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400
600
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Peak
Torque
(mN*m/kg)
Mouse VML model

26. Awasthi et al., Onco Targets Therp 2015
Anti-Fibrotic Treatment Nintedanib
Intracellular tyrosine kinase inhibitor that
targets fibroblast growth factor receptor
(FGFR) 1-4, platelet-derived growth
factor receptor (PDGFR) α/β, vascular
endothelial growth factor receptor
(VEGFR) 1-3, and the Src family of
tyrosine kinases Lck, Lyn, and Flt-3
Pig VML model
Corona et al., Tis Eng A 2020

27.  Anti-fibrotic treatment diminished the VML-
induced change in stiffness
 Non-repaired and non-treated VML muscle
have a 4-7 fold increase in stiffness 1-month
post-injury
Superficial
Deep
Corona et al., Tis Eng A 2020

28.  Greater force deficit in the anti-fibrotic treated
group than the VML non-repaired, non-
treated
 One-month post-VML there is a similar deficit
of ~35% when normalized for muscle size
Corona et al., Tis Eng A 2020

29.  Preservation of directionality of the natural wound healing response
 In many genes, the anti-fibrotic treatment following VML injury consistently affected
expression by attenuating the magnitude of upregulation
 As opposed to completely inhibiting upregulation (or downregulating)
Corona et al., Tis Eng A 2020
Fibrosis Wound healing

30. Increased pathologic fibrosis
An overaccumulation of extracellular matrix deposition during
the repair process
Impaired healing and regeneration
How do we optimize treatment delivery or timing?
Keep this TGFβ independent?
How to integrate with other approaches?

31. Chronic neural
dysfunction
Full maturation of
regenerated or repaired
muscle fibers is
dependent on fiber
innervation

32. Laminin/Neurofilament
persistent
functional deficits
maintained
descending
axons
maintained α-motor neurons,
but persistent axotomy
Corona et al., Mus Nerv 2018
 Retained but limited capacity for reinnervation

33. Pig VML model
 No motor neuron death, but
significant axotomy
 Notably, motor neurons
have retained capacity for
reinnervation
maintained α-motor neurons
 Retained but limited capacity for reinnervation

34. Nuclei (DAPI) / Axons (2H3-SV2 & S100) / NMJ αBTX
 What about innervation
at the fibers?

35. Sorensen et al., J Appl Phys 2021
Secondary enervation and the chronic appearance of
sprouting and poly-innervation are part of the sequela of injury
Contribute to the chronic loss of muscle function post-VML
injuries
The maintenance of NMJ size and complexity indicate the
potential for NMJs to be reinnervated, supportive to retention
of motor neurons
Pre-synaptic (2H3-SV2) / Post-synaptic (αBTX)

36. Post-synaptic NMJ (αBTX) / Axons Pre-synaptic (2H3-SV2) / Nuclei (DAPI) / S100
tSC co-localization
What about the specialized terminal Schwann cell (tSC)?

37. Post-synaptic NMJ (αBTX) /
Axons Pre-synaptic (2H3-SV2) /
Nuclei (DAPI) / S100 (tSC co-
localization)
Hoffman et al., In Review
Delayed increase in tSC
number
Possible pathologic
trophic activity

38. Can the potential to reinnervate
be targeted?
Herceptin: Monoclonal Antibody
designed to target the ErbB2/HER2
receptor in Breast cancer cells.
In skeletal muscle
Neuregulin activates the ErbB/HER
receptors for downstream activation
of nerve development and other
cellular processes
Has shown a paradoxical role in
promoting axon outgrowth following
inhibition
Sorensen et al., In Review

39.  No functional
improvement
 Able to mitigate
secondary denervation
and pathologic NMJ
characteristics
Sorensen et al., In Review
 Although paradoxical,
inhibiting the ErbB2
receptor chronically

40. Chronic neural dysfunction
 Prolonged motor neuron axotomy and secondary denervation may have
considerable impact on the functional capacity
 Optimistically, there is a lot of remaining potential
 How do we optimize treatment delivery or timing?
 How do we optimize the development of regenerative medicine approaches
to support reinnervation, considering an overactive local environment?
 Understand and target supporting aspects of the NMJ like the tSCs, along
with all the other aspects (e.g., satellite cells, FAPS, growth factors, immune
cells) is important to support ongoing potential for stabilization and
reinnervation in efforts to support long-term functional recovery

41. Metabolic maladaptively
Lack of standard metabolic
response locally
Whole-body implications

42. Acute and local destruction of the mitochondrial network that
corresponds to mitochondrial dysfunction
Southern et al., Sci Rep 2019

43. Distance from Injury Site
Southern et al., Sci Rep 2019

44. Greising et al., BMC Muscul Dis 2018 Southern et al., Sci Rep 2019
Injury-induced increase in mitochondrial content in the remaining muscle, but
they have a reduced oxidative capacity both acutely and chronically
S h a m VM L
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3
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0 .6
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Chao et al., J Appl Phys 2019
Pig VML model Mouse VML model

45. Mitochondrial respiration states are impaired across
the respiratory states chronically
McFaline-Figueroa et al, Tis Eng A 2022

46. Electron conductance is slowed in VML-
injured muscle compared to injury naïve
muscle and this is associated with a
greater mitochondrial membrane potential
Impaired carbohydrate lipid oxidation
oxygen consumption (JO2) at different ATP Gibb’s
free energy states for carb vs. fat substrate
McFaline-Figueroa et al, In Review

47. Dalske et al., PLOS 2021
Injury-induced impairment in whole-body metabolism
M
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48. Decreased carbohydrate oxidation and increased lipid oxidation
Due primarily to changes in substrate oxidation and fuel selection
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Raymond-Pope et al., In Review

49. Metabolic maladaptively
Reduced mitochondrial function and enzymatic activity
Chronic metabolic rate and RER reductions with no change in
activity
Can we optimize a treatment?
How to correct mitochondrial function for long term health?
How to integrate with other approaches?

50. 1) Simple example of scale up with a HGF loaded biomaterial
2) Complex approach of scale up with pharmacologic treatment
of fibrosis
3) Pathophysiologic understand of impairments to support
future scale up
a) Neural
b) Metabolic
4)Summary and next steps

51. Summary
 VML injury is a complex problem that has pathologic implications on the
muscle remaining and not just the area of muscle lost
 Function and physiologic understanding across the translational pipeline is
key to progress

53. Summary
 VML injury is a complex problem that has pathologic implications on the
muscle remaining and not just the area of muscle lost
 Function and physiologic understanding across the translational pipeline is
key to progress
Importance and Future Work
 The complexity will require a multi-disciplinary approach that should first
correct the physiology of the muscle
 Need to investigate how to promote a regenerative permissive
environment that balances physiology and improvements in function
 Evaluate optimal timing and duration of treatments alone or combined with
regenerative medicine approaches in efforts to improve long term function
 Novel approaches that can fill the unmet need

54. Collaborators
Jarrod Call, PhD - University of Georgia
Benjamin Corona, MD PhD - Wake Forest University
Dan Garry, MD PhD – University of Minnesota
Mary Garry, PhD – University of Minnesota
Jessica Rivera, MD PhD – Louisiana State University
Gordon Warren, PhD - Georgia State University
Fibralign Corp.
Michael Paukshto, PhD
Tatiana Zaitseva, PhD
Lab: http://smprl.umn.edu/
@GreisingLab
Current Lab Members
Shefali Bijwadia
Angela Bruzina
Braydon Crum
Daniel Hoffman
Mason Lentz
Thomas Lillquist
Peter Nicholson
Emma Pritchard
Christiana Raymond-Pope, PhD
Past Lab Members (Graduates)
Alec Basten (BS, MS)
Rachael Bloxzom (BS)
Kyle Dalske (MS)
Amanda Russell (BS)
Jacob Sorensen, PhD
Matt Borkowski
Chris Rand
Sydney Mensen

55. Thank you for participating!
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