Healing of nerves, blood vessel, muscles, tendon, cartilage and bone
Peripheral nerves
History
Galen 200AD - not possible
Guy deChauliac (1300s) - repaired ends with restoration
Muller/Schwann 1842 - regeneration confirmed
Waller - distal parts degerated
Tinel (1918) - tingling = nerve regeneration vs pain = nerve irritation
Seddon/Woodhall - bridge/cable grafts/primary/secondary repair
Sidney Sunderland(1978) - anatomy of nerves
Anatomy
Cell bodies
motor nerves - anterior horn of the spinal cord
sensory nerve - dorsal root ganglia
Motor nerves terminate at motor end plates in muscle and sensory nerves
terminate in sensory receptors in skin.
When neuronal cell body is injured the entire cell dies
Axonal injury do not result in cell death rather the damaged neurons respond by
regenerating new distal axonal segments
Axonal coverings:
+/-myelin sheath
Schwann cells.
Endoneurium. Resists longitudinal forces
perineurium = nerve fascicles. Ala blood brain barrier. Removal leads to
cessation of nerve function.
epineurium surround groups of perineural bound bundles. The vessels run
in the epineurium. The epineurium is rich in fibroblasts. Thicker around
joints
An inner epineurium surrounds one or more fascicular bundles within
larger peripheral nerves with the outer epineurium surrounding the entire
peripheral nerve
Proteins and other substances required for maintenance of cellular homeostasis
and for signal transmission are synthesized primarily within the cell body. These
substances are then transmitted down the axon through a micro tubular system to
the site where they are required
Blood supply
• Vasa nervorum enter the nerve segmentally.
• Longitudinal vessels run both superficially in the outer epineurium and
deep between the fascicles.
Physiology
Action Potentials
•
Localised potentials: Occur over short distances, decrease over distance,
important at sensory nerve endings and inter-cellular junctions.
• Action potentials: Conducted impulses that do not diminish over time.
• Action potentials in unmyelinated fibres progress at a rate directly
proportional to the size of the fibre.
• In myelinated fibres, AP’s jump from one node of Ranvier the next, a
process called saltatory conduction, that speeds up conduction
tremendously.
Axoplasmic flow
• Bidirectional: Antegrade may be either fast or slow.
• Fast antegrade flow plays a functional role for transport of
neurotransmitter vesicles and plasma membrane constituents.
• Slow antegrade flow is important for structural elements of the
cytoskeleton: micro tubules and filaments and neuro-filaments.
• Retrograde flow is for evacuation of waste products, recycling of cell
products and the relay of information to the cell body.
Nerve injury
4 PHASES OF REGENERATION
Degeneration: The phase of delay: About 1 month
Axon penetration of nerve interface: Crossing the gap
Distal axon regeneration down the endoneurial tube: ~1mm/d
Functional recovery
Degeneration
Distal
Wallerian degeneration - process of fragmentation of the axon distal to
the injury as well as its myelin sheet
Injury causes release of ca2+ ions which mediate activation of proteases
that results in the formation of free radicals which both contribute to tissue
breakdown
As tissue degenerates the Schwann cells proliferate and become
phagocytic
They phagocytize degenerating axonal elements and myelin -> clearing of
distal axonal segments and contribute to the phagocytic process
End result = hollow endoneural sheath with adjacent Schwann cells which
then collapses. By the end of 2-6 weeks, no histological trace of the distal
axon can be found.
Proximal
limited degeneration occurs in a similar fashion (called axonal
degeneration).
Distance of proximal degeneration depends on the severity of the injury
and degrades to the next node of Ranvier
Schwann cells secrete cytokines nerve growth factor(promotes axonal extension)
Macrophages contribute
1. NGF
2. nsulin like GF (IGF promotes axonal elongation)
3. PDGF apolipoprotein E (axonal elongation and myelination)
Changes in cell body after injury
Microscopy
Nucleus swells and cell becomes rounder
Ribosomes increase in number
RER breaks up and moves to the periphery
Marginated ER fragments (Nissle bodies)
These changes are termed Chromatolysis
As changes occur there is a decreased synthesis of neuro transmitter and increased
synth of lipids and protein which are transported to site of injury
At site of injury axons sprout from proximal nerve segment within 24 hrs
They derive from both the cut axonal end and from nodes of Ranvier
Initially unmyelinated and individual axons may produce more than one
sprout
A region of axo-plasmic enlargement known as the growth cone develop
at the tip of the sprouts.
The growth cone includes many intracellular structures including
endoplasmic reticulum, microtubules, microfilaments, large mitochondria
and lysosomes
They include Schwann cells at their periphery
Actin rich filopodia extend out and then contract from the most distal part
of the growth cones in ameboid fashion and out in different directions
until they come into contact with a favourable physical substrate.
Substrates include
Schwann cells
Attachment factors such as fibronectin and laminin
Substances found within endoneural sheaths
Neurotrophic factors released by the denervated structures contribute to the
growth of the axon towards it and applies to both motor and sensory nerves
NGF is one of the factors which contributes to the accelerated and directed
growth of axons
IGF may also have similar role
FGF and IL -1 stimulate Schwann cells proliferation and may be involved along
with ciliary neurotrophic factors
The ultimate amount of axons reaching the end organ is generally less than the pre
injury number and central compensation and re-education must occur to maximize
the final result.
Cell body and proximal nerve hypertrophy
Following injury, the cell body and the proximal axon hypertrophy d/t the accumulation
of gel like amorphous substance containing mucopolysaccaride.
RNA and protein content in the cell body increases
Axoplasmic flow continues for a short while.
Schwannoma formation
Schwann cells and fibroblasts proliferate to form a Schwannoma- a mass of scar tissue.
Axonal sprouting
 After a delay of 24 hours to 4 days, the proximal axon forms a growth cone from
which several axons (filopodia) start to sprout.
 This quiescent phase is a period of protein synthesis by the cell bodies.
 Sprouting occurs both from the growth cone and proximally from several nodes of
Ranvier proximal to the injury.
 The aim of sprouting is for axons to bridge the gap.
 The regenerating unit is guided distally by a combination of forces.
o Contact guidance
o neurotropism are both operative
o chemical and electrical mediators facilitate the filopodia’s search for a distal
remnant. Schwann-cell-insulin-like GF may play a role.
 A large gap or the presence of abundant scarring or FB, will diminish the number of
axons that successfully bridge the gap and enter distal endoneurial tubules.
 Smaller fibres (eg, pain and temperature) seem to be more successful in penetrating
endoneurial tubules.
 When one axonal sprout makes contact with the distal neural elements, it attaches by
fusion of cell membrane. Some degree of contraction occurs pulling the proximal and
distal ends together.
 The new axons are rapidly enveloped by Schwann cells from the proximal nerve end.
 Myelination is determined by the proximal parent nerve.
Regeneration down the distal endoneurial tube
Distal axon regeneration down the neural tube occurs at a variable rate dependent on a
number of factors:
Site: Regeneration is faster proximally than distally. 8 mm/d in the upper arm; 1
mm/d in the hand.
Scar tissue: Retards the rate of progress to about 0.25 mm per day.
Grafts: In non vascularised nerve grafts, 3-4 mm/day.
Trophic factors:
Steroids slow the rate of regeneration;
T3 and nerve growth factor increase it.
When the axon encounters the target organ, other sprouts are triggered to degenerate.
Tinel sign is most useful to determine the rate of axonal regeneration.
Usually there is a delay period of about a month before the Tinel becomes positive.
As axons advance, myelination proceeds in a centrifugal manner.
By 3 weeks after injury, axon regeneration is the predominant feature. If these axons
become trapped in proliferating fibroblastic tissue, neuroma results.
Changes in the motor end plates and muscle
After about 3 months, the motor end plates start to become increasingly distorted due to
connective tissue in-growth. Muscle fibres start to undergo progressive shrinkage. Reinnervation, however, is possible for up to 3 years following injury.
Atrophy and degeneration of end plates and muscle can be retarded by external
stimulation.
Sensory recovery is dependent on the re-innervation of existing sensory corpuscles rather
than on the development of new ones.
There is a variable return of function after end organ re-innervation.
Re-education and rehabilitation improve the degree of functional recovery.
Injury Classification
Severity of injury to the axon can vary and affect the healing response. Two
classifications by seddon(1948) and sunderland(1968)
Seddon:
neuropraxia - conduction block with no actual structural damage to cell,
and axonal regeneration is not required after this injury which is usually a
transient compression of the nerve
axonotmesis - involves damage to internal nerve structures whereas outer
most epineurium remains intact (Sunderland classifies this group according to the
nerve structure that is actually damaged)
neurotmesis - involves all peripheral structures such as laceration of the
nerve and is the most common type leading to surgical intervention
Testing
Sensory
Slow adapting fibers (pulse thruout duration)
static 2PD
Semmes-Weinstein monofilament
Quick adapting fibers (on-off)
vibration
moving 2PD
Threshold test (a single nerve fibre to a group of receptors)
- Semmes-Weinstein monofilament and vibration
- best test for nerve compressions
Innervation density test (density of innervation to an area)
- static and moving 2pd
- best test for acute nerve trauma but not gradual compression
Nerve conduction velocities and latencies
Repair
Number of factors influence the results of nerve repair:
Local factors
1) Injury at Multiple levels: and more severe injuries create defects with significant
scarring between ends esp true in severe ischaemic insult. The regenerating axons
have limited ability to generate the proteases required for scar penetration and
thus branch inresponse to this. GROWTH THRU NERVE GRAFT IS 2-3 MM/D
WHEREAS GROWTH through scar is 0.25mm/d> as severity of injury increases
the chance of injured nerve not finding distal sheath increases.
2) Injury to proximal Nerve - injuries more proximal to involved muscles and
sensory end organs achieve much better results than when structures must
regenerate over long distances. This is partly due to target organ degeneration and
increased homogeneity of distal nerves which lead to less cross innervation .More
proximal muscles tends to require less precise function than distal muscle.
3) Devascularization
4) Poor wound bed - massive crush injury, scarring, hematoma, infection
Patient factors
1) Delayed Repair - if long time passes between injury and repair the sheath
diminishes in size limiting myelin thickness, axonal size and functional result.
Esp true in motor nerves as the motor end plates atrophy and become less
functional with time
2) Increased age - age significant variable affecting results with individuals over 40
achieving much poorer results than those under 40. May involve cortical plasticity
rather than axonal regeneration
Surgical factors
1) surgical skill (alignment, gap, handling), rehabilitation.
Basic principle of repair
1) Careful handling of tissue
2) Limited devascularization of proximal and distal nerve
3) Tension free closure
4) Careful coaptation of nerve ends
Primary vs Secondary Repairs (>1 week)
Primary repair shown to be superior in animals and human studies
Indications for secondary repair
1. crush injury
2. contaminated bed
3. other injuries
Techniques
1. Epineural
2. Group fascicular
3. Individual fascicular
Fascicular repair has not shown to give better results than epineural repair probably as
there is increases scarring generated by intraneural repair.
Fascicular matching - mainly to separate sensory from motor nerves
1. Intraop nerve stimulation
awake patient
proximal stump - map sensory
distal stump - map motor (only work first 1-3 days)
2. Histochemical analysis
Anticholinesterase - myelinated motor and small unmyelinated axons but
not myelinated sensory nerves
Carbonic anhydrase - myelin and axons of myelinated sensory nerves
Proximal end - indefinite period, Distal end - first 9 days
Need to resect nerve ends to histochemistry, 1-2 hrs processing time.
? more useful in late reconstruction
Aftercare
Immobilise for 3 weeks
Splinting positions
Sensory reeducation
Blood vessels
Anatomy
Anatomy varies depending on size of vessel
Blood vessels are composed of following layers or tunics
1) Tunica Intima - consists of a layer of endothelial cells lining the inner surface that
rest on basal lamina and have turnover of 1%/ day beneath endothelium is the
subendothelium consisting of loose areolar tissue that may contain occasional
smooth muscle cells that are both arranged longitudinally
2) Tunica Media - consists chiefly of concentric layers of helically arranged smooth
muscle cells interposed with variable amounts of elastic and reticular fibres
In ateries the intima and media are separated by the internal elastic lamina
composed of elastin and is fenestrated to allow diffusion to nourish vessel wall
A thinner external elastic lamina is often found separating the media from the
outer adventitia
3) Tunica Adventitia -consists principally of longitudinally orientated collagen fibres
collage in the adventitia is type I and in the media is mainly type III
Vaso Vasorum (vessels of Vessels in larger vessels)branch in the adventitia and the
outer part of the media; and provide metabolites to the adventitia and media and the
inner layers are diffused from the lumen
Vessel Injury
Injuries limited to the endothelium can be produced by minor trauma eg
1) surgical dissection of blood vessels
2) Desiccation of the vessel
3) Prolonged spasm
4) The application of a microvascular clamp can cause endothelial loss
When endothelial loss occurs, the endoth is reconstituted by the endothelial cells
that migrate from the edges of the denuded area. A 1cm to 1.5 cm area is
generally completely covered in 7- 10 days
Healing at anastomosis requires healing of all tissue layers
In the anastomotic portion of artery the endoth and internal elastic lamina is lost
exposing the connective tissue elements of the deeper layers. Endothelial damage
can occur over a wider area
Platelets aggregate where subendothelial collagen is exposed. Subendoth micro
fibrils and basement membrane stimulate platelet aggregation as do sutures.
However only a thin layer of platelets may aggregates in the region of a well
formed anastomosis
The carpet of platelets starts to form immediately as the blood starts to flow at the
anastomosis and aggregation peaks several hours after blood flow is to the
anastomotic site. Although some fibrin and red cells are included in the platelet
thrombus significant fibrin is not produced unless there is significant constriction
of the blood vessel or extensive expose of the media. The layers of platelets in the
thrombus is greater in veins than in arteries.
The thickness of platelet carpet increases for the first 4 hours post op and then
gradually decreases in size. The platelets slowly disappear over 3-7 days as
endothelial proliferation increases
Neutrophils are seen at the anastomotic site within hours of a vascular repair and
begin to replace the RBC that is initially seen. Macrophages are seen in large
numbers approx 3 days after the vasc repair and these phagocytic cells contribute
to the reduction in size of the thrombus over the first week.
At 5 days sutures are covered by pseudointima consisting of thrombus fibrin and
leukocytes. Endothelial cell migration across anastomotic site begins several days
after and by 14 days after the sutures are completely covered by endothelium
however the endothelial surface is irregular and take 8 weeks to flatten and
smooth
The endothelial repair is modulated by the matrix on which the cells grow and by
cytokines that stimulate endothelial cell activities. Endothelial cell migration
involves the following
a. Regulated attachment and detachment of cells
b. Contraction of cytoplasmic filaments
c. Changes in the plasticity of the cytoskeleton
d. Regulation of cell to cell communication
Several cytokines influence endothelial cell activity including
FGF
Epidermal growth factor
TGF alpha
IGF I
PDGF
VEGF







Cytokines such as IL1 and TNF alpha may help smooth muscle and collagenases
involved in the damaged matrix. Endothelial cells synthesize some of these
cytokines including BFGF, PDGF,TGF , IGF-Ialthough other cytokines
contribute to the repair process
Healing of deeper vessel layers continue for months at an anastomotic site.
Some medial necrosis is produced by the sutures. Smooth muscle cells and
fibroblasts migrate from adjacent areas and produce collagen and other
connective tissue elements which seal the deeper vessel layers.
Excessive collagen production can lead to intimal thickening and hyperplasia.
Myointimal thickening may occur as early as 10 days after an anastamosis and at
three months the wall thickness in arteries may be doubled.
process is much more active in arteries than in veins.
gradually remodel and decrease in thickness over years
When vein grafts are used in arteries
the endothelium initially sloughs from a large portion of the vessel
At anastomotic site all endothelium is lost but further from anast only partial
endoth loss may occur.
Areas of loss covered by thrombus and then endothelium regenerates over 14 d
The normal media and intima are replaced by a fibromuscular neointima with
long smooth muscle cells , fibroblasts and collagen. The smooth muscle cells have
fewer contractile elements and more synthetic elements than usually seen and
these cells contribute to the synthesis of matrix components. The cellular elements
most likely originate from the native vessels and migrate into the grafts,
Myointimal thickening is most apparent at 14 days and gradually improves with
scar remodelling At 6 mnth macrophages contain lipid in the walls of a vein graft
and the number diminish by 12 months. Vein grafts in the venous side don’t
undergo as many changes as they are not subject to increased forces
Muscle
In response to injury muscle will regenerate or form scar tissue
Skeletal muscle more often responds to injury by regeneration where as smooth
muscle more often through scar production
Regeneration of cardiac muscle will occur when individual fibres have been
damaged with preservation of endomysium,
For regeneration occur the muscle components must remain in close
approximation
Thus if damage is extreme as in severe ischaemia or infarction then more likely to
heal with scar formation
Anatomy
Skeletal muscle consist of multiple elongated cells packed into fibre bundles. The
cells have multiple nuclei and a large amount of cytoplasm containing myofibrils.
Each cell is circumscribed by its sarcolemma which includes the cellular
membrane and an endomysium consisting of connective tissue elements. Bundles
of muscle fibres known is fascicles are circumscribed by a perimysium. The
muscle as a whole is surrounded by a epimysium
 The vascular and neural supply courses within the sarcolemma. With any injury
local muscle degeneration results from damage to these small nerve fibres even if
the primary nerve supply to muscle remains intact
Muscle Injury
When muscle is lacerated the cellular elements retract leaving the sarcolemma empty for
a short distance. Three distinct areas have been identified in the zone of injury
1) uninjured zone prox to injury
2) central area of wound including clot
3) Intermediate surviving zone including sarcolemmal membranes
The dense clot at the retracted ends of the muscle fibres appear as a cap over the
injured muscle which includes fibrin and fibronectin released from he cellular
elements of the plasma.
The matrix facilitates the migration of inflammatory cells and then fibroblasts into
the injured area
During the first 2 days the injured monocytes seal with the formation of new
membrane and neutrophils migrate into the injured muscle.
Day 3 - the basal laminae of empty sarcolemmal sheaths are lined by
macrophages which are actively phagocytizing degenerating cellular elements and
debris. During this period revascularization of the injured area is also occurring
under the influence of bFGF and TGF b
Day 5 the inflammatory cells start to diminish and by day 10 are rarely seen and
fibroblastic proliferation is extensive. Collagen is initially type III but within days
more type I develops. The collagen is located in the granulation tissue and clot at
the injury site and in the endomysium
Regeneration process
The source of regenerating muscular elements are small cells that adhere tho the
basal lamina of the of the sarcolemma and lie next to the larger cells. Unlike the
elongated multinucleated cells which have a prominent cytoplasm containing
myofibrils these satellite cells have large nuclei and scant cytoplasm
o Satellite cells found only in skeletal muscle and thus explains their
regenerating capacity and theses cells are the only cells in the muscle that
are capable of mitosis
o Provide 5 % of the nuclei found in muscle
o
After injury satellite cells become myoblasts and through repeated mitosis
repopulate the injured area with muscular elements
o This process is stimulated by FGF,IGF and TGFB.
o After many cells are produced they fuse and form multinucleated cells
which then produce contractile proteins
Phases of regeneration
Myoblastic stage
o Myoblasts are predom dividing
o Peaks at 48 - 72 hrs after injury
o By day 4-6 sarcolemma tubes are lined by myo blast
Myotubular stage
o
Occurs when fibronectin concentrations diminish and myoblasts fuse and become
synthetic
o The intracellular myofibrils become longer and longer as the as more actin and
myosin
o Muscle cells start to extend from the sarcolemmal tube and is evident for 4-7 days
after the injury
o By day 7 cross striations appear
o By day 14 they after started to bridge the gap created by the injury but at this
stage the muscle fibres are still attenuated and disorganized
o Day 21 -myofibrils are deeply staining with abundant cross striations and clear
sarcolemma is formed
Maturational stage
o Final architecture is restored
o Cross striations become more prominent in the myofibrils and myofibrils
gradually increase in thickness
o Tension is required for myofibrillary orientation to occur
o Reinnervation Takes place in the final stage of healing
IN severe injury ischaemia results and the satellite cells required for the regenerative
process do not survive thus scar results
Complete muscle regeneration is also not possible if wide separation between ends exist
Management and mobilization
o increased neovascularization and allows for more rapid regeneration of normal
architecture and function in muscle
o Mobilization can also resulting further muscle disruption resulting in increased
granulation tissue formation and fibrosis
o In a rat model after 5 days of immobilization sufficient tensile strength was
regained to prevent subsequent re-rupture when mobilization was commenced
o Mobilization allow better regeneration of muscle and resorption of connective
tissue synthesized in the injured area after the injury
o Prolonged immobilization results in increased amounts of collagen and less
organized muscle regeneration